Nucleotides and nucleosides and methods for their use in DNA sequencing

ABSTRACT

The present invention relates generally to labeled and unlabled cleavable terminating groups and methods for DNA sequencing and other types of DNA analysis. More particularly, the invention relates in part to nucleotides and nucleosides with chemically cleavable, photocleavable, enzymatically cleavable, or non-photocleavable groups and methods for their use in DNA sequencing and its application in biomedical research.

The present application is a continuation application of U.S. patentapplication Ser. No. 13/929,305, filed Jun. 27, 2013, which is adivisional application of U.S. patent application Ser. No. 13/406,934filed Feb. 28, 2012, issued as U.S. Pat. No. 8,497,360, which is adivisional application of U.S. patent application Ser. No. 12/483,080filed Jun. 11, 2009, issued as U.S. Pat. No. 8,148,503, which claims thepriority to U.S. Provisional Application Nos. 61/060,795 and 61/184,779,filed Jun. 11, 2008 and Jun. 5, 2009, respectively, the entire contentsof which are each incorporated herein by reference.

BACKGROUND OF THE INVENTION

I. Field of the Invention

The present invention relates generally to compositions and methods forDNA sequencing and other types of DNA analysis. More particularly, theinvention relates in part to nucleotides and nucleosides with chemicallycleavable, photocleavable, enzymatically cleavable, ornon-photocleavable groups and methods for their use in a number of DNAsequencing methods and their applications in biomedical research.

II. Description of Related Art

Methods for rapidly sequencing DNA have become needed for analyzingdiseases and mutations in the population and developing therapies.Commonly observed forms of human sequence variation are singlenucleotide polymorphisms (SNPs), which occur in approximately 1-in-300to 1-in-1000 base pairs of genomic sequence and structural variants(SVs) including block substitutions, insertion/deletions, inversions,segmental duplications, and copy number variants. Structural variantscan accounted for 22% of all variable events and more variant bases thanthose contributed by SNPs (Levy et al., 2007, which is incorporatedherein by reference.). This finding is consistent with that of Scherer,Hurles, and colleagues who analyzed 270 individuals usingmicroarray-based methods (Redon et al. 2006, which is incorporatedherein by reference). Building upon the complete sequence of the humangenome, efforts are underway to identify the underlying genetic link tocommon diseases by SNP mapping or direct association. Technologydevelopments focused on rapid, high-throughput, and low cost DNAsequencing would facilitate the understanding and use of geneticinformation, such as SNPs, in applied medicine.

In general, 10%-to-15% of SNPs will affect protein function by alteringspecific amino acid residues, will affect the proper processing of genesby changing splicing mechanisms, or will affect the normal level ofexpression of the gene or protein by varying regulatory mechanisms. SVsmay also play an important role in human biology and disease (Iafrate etal., 2004; Sebat et al., 2004; Tuzun et al., 2005; Stranger et al.,2007, which are incorporated herein by reference). It is envisioned thatthe identification of informative SNPs and SVs will lead to moreaccurate diagnosis of inherited disease, better prognosis of risksusceptibilities, or identity of sporadic mutations in tissue. Oneapplication of an individual's SNP and SV profile would be tosignificantly delay the onset or progression of disease withprophylactic drug therapies. Moreover, an SNP and SV profile of drugmetabolizing genes could be used to prescribe a specific drug regimen toprovide safer and more efficacious results. To accomplish theseambitious goals, genome sequencing will move into the resequencing phasewith the potential of partial sequencing of a large majority of thepopulation, which would involve sequencing specific regions in parallel,which are distributed throughout the human genome to obtain the SNP andSV profile for a given complex disease.

Sequence variations underlying most common diseases are likely toinvolve multiple SNPs, SVs, and a number of combinations thereof, whichare dispersed throughout associated genes and exist in low frequency.Thus, DNA sequencing technologies that employ strategies for de novosequencing are more likely to detect and/or discover these rare, widelydispersed variants than technologies targeting only known SNPs.

Traditionally, DNA sequencing has been accomplished by the “Sanger” or“dideoxy” method, which involves the chain termination of DNA synthesisby the incorporation of 2′,3′-dideoxynucleotides (ddNTPs) using DNApolymerase (Sanger et al., 1997, which is incorporated herein byreference). The reaction also includes the natural 2′-deoxynucleotides(dNTPs), which extend the DNA chain by DNA synthesis. Balancedappropriately, competition between chain extension and chain terminationresults in the generation of a set of nested DNA fragments, which areuniformly distributed over thousands of bases and differ in size as basepair increments. Electrophoresis is used to resolve the nested DNAfragments by their respective size. The ratio of dNTP/ddNTP in thesequencing reaction determines the frequency of chain termination, andhence the distribution of lengths of terminated chains. The fragmentsare then detected via the prior attachment of four differentfluorophores to the four bases of DNA (i.e., A, C, G, and T), whichfluoresce their respective colors when irradiated with a suitable lasersource. Currently, Sanger sequencing has been the most widely usedmethod for discovery of SNPs by direct PCR sequencing (Gibbs et al.,1989, which is incorporated herein by reference) or genomic sequencing(Hunkapiller et al., 1991; International Human Genome SequencingConsortium, 2001, which are incorporated herein by reference).

Advantages of next-generation sequencing (NGS) technologies include theability to produce an enormous volume of data cheaply, in some cases inexcess of a hundred million short sequence reads per instrument run.Many of these approaches are commonly referred to assequencing-by-synthesis (SBS), which does not clearly delineate thedifferent mechanics of sequencing DNA (Metzker, 2005, which isincorporated herein by reference). Here, the DNA polymerase-dependentstrategies are classified as cyclic reversible termination (CRT), singlenucleotide addition (SNA, e.g., pyrosequencing), and real-timesequencing. An approach whereby DNA polymerase is replaced by DNA ligaseis referred to as sequencing-by-ligation (SBL).

There is a great need for developing new sequencing technologies, withpotential applications spanning diverse research sectors includingcomparative genomics and evolution, forensics, epidemiology, and appliedmedicine for diagnostics and therapeutics. Current sequencingtechnologies are often too expensive, labor intensive, and timeconsuming for broad application in human sequence variation studies.Genome center cost is calculated on the basis of dollars per 1,000 Q₂₀bases and can be generally divided into the categories ofinstrumentation, personnel, reagents and materials, and overheadexpenses. Currently, these centers are operating at less than one dollarper 1,000 Q₂₀ bases with at least 50% of the cost resulting from DNAsequencing instrumentation alone. Developments in novel detectionmethods, miniaturization in instrumentation, microfluidic separationtechnologies, and an increase in the number of assays per run will mostlikely have the biggest impact on reducing cost.

SUMMARY OF THE INVENTION

In some aspects, the present disclosure provides novel compounds andcompositions that are useful in efficient sequencing of genomicinformation in high throughput sequencing reactions. In another aspect,reagents and combinations of reagents that can efficiently andaffordably provide genomic information are provided. In further aspects,the present invention provides libraries and arrays of reagents fordiagnostic methods and for developing targeted therapeutics forindividuals.

In some aspects, the present disclosure provides new compounds that maybe used in DNA sequencing. For example, in some embodiments, theinvention provides compounds of the formula:

wherein:

-   -   Z is —O—, —S—, —NH—, —OC(O)O—, —NHC(O)O—, —OC(O)NH— or        —NHC(O)NH—;    -   R₁ is hydroxy, monophosphate, diphosphate, triphosphate or        polyphosphate;    -   R₂ is hydrogen or hydroxy;    -   R₃ and R₄ are each independently:        -   hydrogen, hydroxy, halo or amino; or        -   alkyl_((C≦12)), alkenyl_((C≦12)), alkynyl_((C≦12)),            aryl_((C≦12)), aralkyl_((C≦12)), hetero-aryl_((C≦12)),            heteroaralkyl_((C≦12)), alkoxy_((C≦12)), aryloxy_((C≦12)),            aralkoxy_((C≦12)), heteroaryloxy_((C≦12)),            heteroaralkoxy_((C≦12)), alkyl-amino_((C≦12)),            dialkylamino_((C≦12)), arylamino_((C≦12)),            aralkylamino_((C≦12)), or a substituted version of any of            these groups; or    -   R₅, R₆, R₇, R₈ and R₉ are each independently:        -   hydrogen, hydroxy, halo, amino, nitro, cyano or mercapto;        -   alkyl_((C≦12)), alkenyl_((C≦12)), alkynyl_((C≦12)),            aryl_((C≦12)), aralkyl_((C≦12)), hetero-aryl_((C≦12)),            heteroaralkyl_((C≦12)), alkenyl-acyl_((C≦12)),            alkoxy_((C≦12)), alkenyl-oxy_((C≦12)), alkynyloxy_((C≦12)),            aryloxy_((C≦12)), aralkoxy_((C≦12)),            hetero-aryloxy_((C≦12)), heteroaralkoxy_((C≦12)),            acyloxy_((C≦12)), alkylamino_((C≦12)),            dialkylamino_((C≦12)), alkoxyamino_((C≦12)),            alkenylamino_((C≦12)), alkynyl-amino_((C≦12)),            arylamino_((C≦12)), aralkylamino_((C≦12)),            heteroaryl-amino_((C≦12)), heteroaralkylamino_((C≦12)),            alkylsulfonylamino_((C≦12)), amido_((C≦12)),            alkylthio_((C≦12)), alkenylthio_((C≦12)),            alkynylthio_((C≦12)), aryl-thio_((C≦12)),            aralkylthio_((C≦12)), heteroarylthio_((C≦12)),            heteroaralkyl-thio_((C≦12)), acylthio_((C≦12)),            thioacyl_((C≦12)), alkylsulfonyl_((C≦12)),            aryl-sulfonyl_((C≦12)), alkylammonium_((C≦12)),            alkylsulfonium_((C≦12)), alkyl-silyl_((C≦12)), or a            substituted version of any of these groups;        -   a group of formula:

-   -   -   -   wherein                -   X is                -    —O—, —S—, or —NH—; or                -    alkanediyl_((C≦12)), alkenediyl_((C≦12)),                    alkynediyl_((C≦12)), arenediyl_((C≦12)),                    heteroarenediyl_((C≦12)), or a substituted version                    of any of these groups;                -   Y is —O—, —NH—, alkanediyl_((C≦12)) or substituted                    alkanediyl_((C≦12));                -   n is an integer from 0-12; and                -   m is an integer from 0-12; or

        -   a -linker-reporter;            or a salt, esters, hydrates, solvates, tautomers, prodrugs,            or optical isomers thereof.

In some aspects the invention provides a compound of formula:

wherein:

-   -   Z is —O—, —S—, —NH—, —OC(O)O—, —NHC(O)O—, —OC(O)NH— or        —NHC(O)NH—;    -   R₁ is hydroxy, monophosphate, diphosphate, triphosphate or        polyphosphate;    -   R₂ is hydrogen or hydroxy;    -   R₃ and R₄ are each independently:        -   hydrogen, hydroxy, halo or amino; or        -   alkyl_((C≦12)), alkenyl_((C≦12)), alkynyl_((C≦12)),            aryl_((C≦12)), aralkyl_((C≦12)), hetero-aryl_((C≦12)),            heteroaralkyl_((C≦12)), alkoxy_((C≦12)), aryloxy_((C≦12)),            aralkoxy_((C≦12)), heteroaryloxy_((C≦12)),            heteroaralkoxy_((C≦12)), alkyl-amino_((C≦12)),            dialkylamino_((C≦12)), arylamino_((C≦12)),            aralkylamino_((C≦12)), or a substituted version of any of            these groups;    -   R₅, R₆, R₇ and R₈ are each independently:        -   hydrogen, hydroxy, halo, amino, nitro, cyano or mercapto; or        -   alkyl_((C≦12)), alkenyl_((C≦12)), alkynyl_((C≦12)),            aryl_((C≦12)), aralkyl_((C≦12)), hetero-aryl_((C≦12)),            heteroaralkyl_((C≦12)), acyl_((C≦12)), alkoxy_((C≦12)),            alkenyl-oxy_((C≦12)), alkynyloxy_((C≦12)), aryloxy_((C≦12)),            aralkoxy_((C≦12)), hetero-aryloxy_((C≦12)),            heteroaralkoxy_((C≦12)), acyloxy_((C≦12)),            alkylamino_((C≦12)), dialkylamino_((C≦12)),            alkoxyamino_((C≦12)), alkenylamino_((C≦12)),            alkynyl-amino_((C≦12)), arylamino_((C≦12)),            aralkylamino_((C≦12)), heteroaryl-amino_((C≦12)),            heteroaralkylamino_((C≦12)), alkylsulfonylamino_((C≦12)),            amido_((C≦12)), alkylthio_((c≦12)), alkenylthio_((C≦12)),            alkynylthio_((C≦12)), aryl-thio_((C≦12)),            aralkylthio_((C≦12)), heteroarylthio_((C≦12)),            heteroaralkyl-thio_((C≦12)), acylthio_((C≦12)),            thioacyl_((C≦12)), alkylsulfonyl_((C≦12)),            aryl-sulfonyl_((C≦12)), alkylammonium_((C≦12)),            alkylsulfonium_((C≦12)), alkyl-silyl_((C≦12)), or a            substituted version of any of these groups; or        -   a group of formula:

-   -   -   -   wherein                -   X is                -    —O—, —S—, or —NH—; or                -    alkanediyl_((C≦12)), alkenediyl_((C≦12)),                    alkynediyl_((C≦12)), arenediyl_((C≦12)),                    heteroarenediyl_((C≦12)), or a substituted version                    of any of these groups;                -   Y is —O—, —NH—, alkanediyl_((C≦12)) or substituted                    alkanediyl_((C≦12));                -   n is an integer from 0-12; and                -   m is an integer from 0-12; or

        -   a -linker-reporter; and

    -   R₉ is alkyl_((C≦12)), aryl_((C≦12)) or a substituted version of        either of these groups;        or a salt, esters, hydrates, solvates, tautomers, prodrugs, or        optical isomers thereof.

In some embodiments, the compound is further defined as a compound offormula A. In some embodiments, the compound is of formula B. In someembodiments, the compound is further defined as a compound of formula C.In some embodiments, the compound is of formula D. In some embodiments Zis —O—. In some embodiments R₁ is hydroxy. In some embodiments, R₁ is amonophosphate. In some embodiments R₁ is a diphosphate. In someembodiments R₁ is a triphosphate. In some embodiments R₁ is apolyphosphate. In some embodiments R₂ is hydrogen. In some embodimentsR₂ is hydroxy. In some embodiments R₃ is hydrogen. In some embodimentsR₃ is alkyl_((C≦12)) or a substituted version thereof. In someembodiments R₃ is alkyl_((C≦8)). In some embodiments R₃ isalkyl_((C≦6)). In some embodiments R₃ is selected from the groupconsisting of methyl, ethyl, n-propyl, isopropyl and tert-butyl. In someembodiments R₃ is methyl. In some embodiments R₃ is alkyl_((C2-6)). Insome embodiments R₃ is alkyl_((C3-5)). In some embodiments R₃ isisopropyl. In some embodiments R₃ is tert-butyl. In some embodiments R₄is hydrogen. In some embodiments R₄ is alkyl_((C≦12)) or a substitutedversion thereof. In some embodiments R₄ is alkyl_((C≦8)). In someembodiments R₄ is alkyl_((C≦6)). In some embodiments R₄ is selected fromthe group consisting of methyl, ethyl, n-propyl, isopropyl andtert-butyl. In some embodiments R₄ is methyl. In some embodiments R₄ isalkyl_((C2-6)). In some embodiments R₄ is alkyl_((C3-5)). In someembodiments R₄ is isopropyl. In some embodiments R₄ is tert-butyl. Insome embodiments R₅ is hydrogen. In some embodiments R₅ is cyano. Insome embodiments R₅ is alkoxy_((C≦12)) or a substituted version thereof.In some embodiments R₅ is alkoxy_((C≦8)). In some embodiments R₅ isalkoxy_((C≦6)). In some embodiments R₅ is alkoxy_((C≦3)). In someembodiments R₅ is methoxy. In some embodiments R₅ is a group of formula:

wherein X is —O—, —S—, or —NH—; or alkanediyl_((C≦12)),alkenediyl_((C≦12)), alkynediyl_((C≦12)), arenediyl_((C≦12)),heteroarenediyl_((C≦12)), or a substituted version of any of thesegroups; and n is an integer from 0-12.

In some embodiments X is alkynediyl_((C≦12)). In some embodiments X isalkynediyl_((C2-8)). In some embodiments X is —C≡C—. In some embodimentsn is zero. In some embodiments R₅ is a group of formula:

wherein X is —O—, —S—, or —NH—; or alkanediyl_((C≦12)),alkenediyl_((C≦12)), alkynediyl_((C≦12)), arenediyl_((C≦12)),heteroarenediyl_((C≦12)), or a substituted version of any of thesegroups; Y is —O—, —NH—, alkanediyl_((C≦12)) or substitutedalkanediyl_((C≦12)); n is an integer from 0-12; and m is an integer from0-12. In some embodiments X is alkynediyl_((C≦12)). In some embodimentsX is alkynediyl_((C2-8)). In some embodiments X is —C≡C—. In someembodiments Y is —CH₂—. In some embodiments n is zero. In someembodiments m is zero. In some embodiments R₆ is a -linker-reporter. Insome embodiments the linker is:

wherein X is —O—, —S—, or —NH—; or alkanediyl_((C≦12)),alkenediyl_((C≦12)), alkynediyl_((C≦12)), arenediyl_((C≦12)),heteroarenediyl_((C≦12)), or a substituted version of any of thesegroups; and n is an integer from 0-12. In some embodiments X isalkynediyl_((C≦12)). In some embodiments X is alkynediyl_((C2-8)). Insome embodiments X is —C≡C—. In some embodiments n is zero. In someembodiments the linker is:

wherein X is —O—, —S—, or —NH—; or alkanediyl_((C≦12)),alkenediyl_((C≦12)), alkynediyl_((C≦12)), arenediyl_((C≦12)),heteroarenediyl_((C≦12)), or a substituted version of any of thesegroups; Y is —O—, —NH—, alkanediyl_((C≦12)) or substitutedalkanediyl_((C≦12)); n is an integer from 0-12; and m is an integer from0-12. In some embodiments X is alkynediyl_((C≦12)). In some embodimentsX is alkynediyl_((C2-8)). In some embodiments X is —C≡C—. In someembodiments Y is —CH₂—. In some embodiments n is zero. In someembodiments m is zero. In some embodiments the linker is:

wherein X is —O—, —S—, or —NH—; or alkanediyl_((C≦12)),alkenediyl_((C≦12)), alkynediyl_((C≦12)), arenediyl_((C≦12)),heteroarenediyl_((C≦12)), or a substituted version of any of thesegroups; Y is —O—, —NH—, alkanediyl_((C≦12)) or substitutedalkanediyl_((C≦12)); n is an integer from 0-12; and m is an integer from0-12. In some embodiments X is alkynediyl_((C≦12)). In some embodimentsX is alkynediyl_((C2-8)). In some embodiments X is —C≡C—. In someembodiments Y is —CH₂—. In some embodiments n is zero. In someembodiments m is zero. In some embodiments the reporter is based on adye, wherein the dye is zanthene, fluorescein, rhodamine, BODIPY,cyanine, coumarin, pyrene, phthalocyanine, phycobiliprotein, ALEXAFLUOR® 350, ALEXA FLUOR® 405, ALEXA FLUOR® 430, ALEXA FLUOR® 488, ALEXAFLUOR® 514, ALEXA FLUOR® 532, ALEXA FLUOR® 546, ALEXA FLUOR® 555, ALEXAFLUOR® 568, ALEXA FLUOR® 568, ALEXA FLUOR® 594, ALEXA FLUOR® 610, ALEXAFLUOR® 633, ALEXA FLUOR® 647, ALEXA FLUOR® 660, ALEXA FLUOR® 680, ALEXAFLUOR® 700, ALEXA FLUOR® 750, or a squaraine dye. In some embodimentsthe reporter is:

In some embodiments R₆ is hydrogen. In some embodiments R₆ isalkoxy_((C≦12)) or a substituted version thereof. In some embodiments R₆is alkoxy_((C≦8)). In some embodiments R₆ is alkoxy_((C≦6)). In someembodiments R₆ is alkoxy_((C≦3)). In some embodiments R₆ is methoxy. Insome embodiments R₆ is a group of formula:

wherein X is —O—, —S—, or —NH—; or alkanediyl_((C≦12)),alkenediyl_((C≦12)), alkynediyl_((C≦12)), arenediyl_((C≦12)),heteroarenediyl_((C≦12)), or a substituted version of any of thesegroups; and n is an integer from 0-12. In some embodiments X isalkynediyl_((C≦12)). In some embodiments X is alkynediyl_((C2-8)). Insome embodiments X is —C≡C—. In some embodiments n is zero. In someembodiments R₆ is a group of formula:

wherein X is —O—, —S—, or —NH—; or alkanediyl_((C≦12)),alkenediyl_((C≦12)), alkynediyl_((C≦12)), arenediyl_((C≦12)),heteroarenediyl_((C≦12)), or a substituted version of any of thesegroups; Y is —O—, —NH—, alkanediyl_((C≦12)) or substitutedalkanediyl_((C≦12)); n is an integer from 0-12; and m is an integer from0-12. In some embodiments X is alkynediyl_((C≦12)). In some embodimentsX is alkynediyl_((C2-8)). In some embodiments X is —C≡C—. In someembodiments Y is —CH₂—. In some embodiments n is zero. In someembodiments m is zero. In some embodiments R₆ is a -linker-reporter. Insome embodiments the linker is:

wherein X is —O—, —S—, or —NH—; or alkanediyl_((C≦12)),alkenediyl_((C≦12)), alkynediyl_((C≦12)), arenediyl_((C≦12)),heteroarenediyl_((C≦12)), or a substituted version of any of thesegroups; and n is an integer from 0-12. In some embodiments X isalkynediyl_((C≦12)). In some embodiments X is alkynediyl_((C2-8)). Insome embodiments X is —C≡C—. In some embodiments n is zero. In someembodiments the linker is:

wherein X is —O—, —S—, or —NH—; or alkanediyl_((C≦12)),alkenediyl_((C≦12)), alkynediyl_((C≦12)), arenediyl_((C≦12)),heteroarenediyl_((C≦12)), or a substituted version of any of thesegroups; Y is —O—, —NH—, alkanediyl_((C≦12)) or substitutedalkanediyl_((C≦12)); n is an integer from 0-12; and m is an integer from0-12. In some embodiments X is alkynediyl_((C≦12)). In some embodimentsX is alkynediyl_((C2-8)). In some embodiments X is —C≡C—. In someembodiments Y is —CH₂—. In some embodiments n is zero. In someembodiments m is zero. In some embodiments the linker is:

wherein X is —O—, —S—, or —NH—; or alkanediyl_((C≦12)),alkenediyl_((C≦12)), alkynediyl_((C≦12)), arenediyl_((C≦12)),heteroarenediyl_((C≦12)), or a substituted version of any of thesegroups; Y is —O—, —NH—, alkanediyl_((C≦12)) or substitutedalkanediyl_((C≦12)); n is an integer from 0-12; and m is an integer from0-12. In some embodiments X is alkynediyl_((C≦12)). In some embodimentsX is alkynediyl_((C2-8)). In some embodiments X is —C≡C—. In someembodiments Y is —CH₂—. In some embodiments n is zero. In someembodiments m is zero. In some embodiments the reporter is based on adye, wherein the dye is zanthene, fluorescein, rhodamine, BODIPY,cyanine, coumarin, pyrene, phthalocyanine, phycobiliprotein, ALEXAFLUOR® 350, ALEXA FLUOR® 405, ALEXA FLUOR® 430, ALEXA FLUOR® 488, ALEXAFLUOR® 514, ALEXA FLUOR® 532, ALEXA FLUOR® 546, ALEXA FLUOR® 555, ALEXAFLUOR® 568, ALEXA FLUOR® 568, ALEXA FLUOR® 594, ALEXA FLUOR® 610, ALEXAFLUOR® 633, ALEXA FLUOR® 647, ALEXA FLUOR® 660, ALEXA FLUOR® 680, ALEXAFLUOR® 700, ALEXA FLUOR® 750, or a squaraine dye. In some embodimentsthe reporter is:

In some embodiments R₇ is hydrogen. In some embodiments R₈ is hydrogen.In some embodiments R₈ is nitro. In some embodiments R₈ isalkyl_((C≦12)) or a substituted version thereof. In some embodiments R₈is alkyl_((C≦8)). In some embodiments R₈ is alkyl_((C≦6)). In someembodiments R₈ is alkyl_((C≦3)). In some embodiments R₈ is methyl. Insome embodiments R₉ is hydrogen. In some embodiments R₉ is nitro. Insome embodiments R₉ is alkyl_((C≦12)) or a substituted version thereof.In some embodiments R₉ is alkyl_((C≦8)). In some embodiments R₉ isalkyl_((C≦6)). In some embodiments R₉ is selected from the groupconsisting of methyl, ethyl, n-propyl, isopropyl and tert-butyl. In someembodiments R₉ is methyl. In some embodiments R₉ is alkyl_((C2-6)). Insome embodiments R₉ is alkyl_((C3-5)). In some embodiments R₉ isisopropyl. In some embodiments R₉ is tert-butyl. In some embodiments R₉is aryl_((C≦12)) or a substituted version thereof. In some embodimentsR₉ is aryl_((C≦8)). In some embodiments R₉ is phenyl.

In some embodiments, the invention provides a compound of the formula:

In some embodiments the compound is a salt of a formula above. In someembodiments, the compound is a 50:50 R:S-at-the-α-carbon-mixture ofdiastereomers of any of the formulas above, or salts thereof, that havea stereocenter at the α-carbon. In some embodiments, the compound ispredominantly one diastereomer substantially free from other opticalisomers thereof. For example, in some embodiments the present disclosureprovides any of the following diastereomers, or salts thereof,substantially free from other optical isomers thereof:

In some embodiments the salt of any of the formulas or diastereomersabove comprises a monovalent cation. In some embodiments the monovalentcation is lithium. In some embodiments the monovalent cation is sodium.In some embodiments the monovalent cation is potassium. In someembodiments the salt comprises a divalent cation. In some embodimentsthe divalent cation is calcium, magnesium or manganese(II). In someembodiments the salt comprises a cation selected from the groupconsisting of ammonium, tri-n-butyl ammonium and (HOCH₂)₃CNH₃ ⁺.

In another aspect the invention provides a method of sequencing a targetnucleic acid comprising the following steps:

-   -   (i) attaching the 5′-end of a primer to a solid surface;    -   (ii) hybridizing a target nucleic acid to the primer attached to        the solid surface;    -   (iii) adding a compound according to any of the claims, with the        proviso that where more than one type of base is present, each        base is attached to a different reporter group;    -   (iv) adding a nucleic acid replicating enzyme to the hybridized        primer/target nucleic acid complex to incorporate the        composition of step (iii) into the growing primer strand,        wherein the incorporated composition of step (iii) terminates        the polymerase reaction at an efficiency of between about 70% to        about 100%;    -   (v) washing the solid surface to remove unincorporated        components;    -   (vi) detecting the incorporated reporter group to identify the        incorporated composition of step (iii);    -   (vii) a cleavage step to remove the terminating moiety resulting        in an extended primer with naturally-occurring bases;    -   (viii) washing the solid surface to remove the cleaved        terminating group; and    -   (ix) repeating steps (iii) through (viii) one or more times to        identify the plurality of bases in the target nucleic acid.

In another aspect the invention provides a method of sequencing a targetnucleic acid comprising the following steps:

-   -   (i) attaching the 5′-end of a target nucleic acid to a solid        surface;    -   (ii) hybridizing a primer to the target nucleic acid attached to        the solid surface;    -   (iii) adding a compound according to any of the claims, with the        proviso that where more than one type of base is present, each        base is attached to a different reporter group;    -   (iv) adding a nucleic acid replicating enzyme to the hybridized        primer/target nucleic acid complex to incorporate the        composition of step (iii) into the growing primer strand,        wherein the incorporated composition of step (iii) terminates        the polymerase reaction at an efficiency of between about 70% to        about 100%;    -   (v) washing the solid surface to remove unincorporated        components;    -   (vi) detecting the incorporated reporter group to identify the        incorporated composition of step (iii);    -   (vii) optionally adding one or more chemical compounds to        permanently cap unextended primers;    -   (viii) a cleavage step to remove the terminating moiety        resulting in an extended primer with naturally-occurring bases;    -   (ix) washing the solid surface to remove the cleaved terminating        group; and    -   (x) repeating steps (iii) through (ix) one or more times to        identify the plurality of bases in the target nucleic acid.

In some embodiments the compound is incorporated by a nucleic acidreplicating enzyme that is a DNA polymerase. In some embodiments the DNApolymerase is selected from the group consisting of Taq DNA polymerase,Klenow(exo-) DNA polymerase, Bst DNA polymerase, VENT® (exo-) DNApolymerase (DNA polymerase A cloned from Thermococcus litoralis andcontaining the D141A and E143A mutations), Pfu(exo-) DNA polymerase, andDEEPVENT™ (exo-) DNA polymerase (DNA polymerase A, cloned from thePyrococcus species GB-D, and containing the D141A and E143A mutations).In some embodiments the DNA polymerase is selected from the groupconsisting of AMPLITAQ® DNA polymerase, FS (Taq DNA polymerase thatcontains the G46D and F667Y mutations), THERMOSEQUENASE™ DNA polymerase(Taq DNA polymerase that contains the F667Y mutation), THERMOSEQUENASE™II DNA polymerase (blend of THERMOSEQUENASE™ DNA polymerase and T.acidophilum pyrophosphatase), THERMINATOR™ DNA polymerase (DNApolymerase A, cloned from the Thermococcus species 9° N-7 and containingthe D141A, E143A and A485L mutations), THERMINATOR™ II DNA polymerase(THERMINATOR™ DNA polymerase that contains the additional Y409Vmutation), and VENT® (exo-) A488L DNA polymerase (VENT® (exo-) DNApolymerase that contains the A488L mutation). In some embodiments thecleavage of the terminating moiety is a chemical cleavage, aphoto-cleavage, electrochemical or an enzymatic cleavage. In someembodiments the chemical cleavage is performed using a catalyst orstoichiometric reagent. In some embodiments the catalyst homogeneous orheterogeneous. In some embodiments the heterogeneous catalyst comprisesPalladium. In some embodiments the homogeneous catalyst comprisesPalladium. In some embodiments 85% to 100% of the photocleavableterminating moieties are removed by means of the photo-cleavage. In someembodiments the photo-cleavage is performed using a wavelength of lightranging between 300 nm to 400 nm. In some embodiments 85% to 100% of thephotocleavable terminating moieties are removed by means of thephoto-cleavage. In some embodiments the invention provides a method ofperforming Sanger or Sanger-type sequencing using a compound disclosedherein. In some embodiments the invention provides a method ofperforming pyrosequencing or pyrosequencing-type sequencing using acompound disclosed herein.

Non-limiting examples of compounds provided by this invention includethe compounds according to the formulas shown below. In certainembodiments, these compounds are substantially free from other opticalisomers thereof.

WW# Chemical Name Diastereomer 1p129 N⁶-(2-nitrobenzyl)-2′-dATP 2p108O⁶-(2-nitrobenzyl)-2′-dGTP 2p143 O⁶-(α-methyl-2-nitrobenzyl)-2′-dGTPmixture 2p148 5-(α-isopropyl-2-nitrobenzyloxy)methyl-2′-dUTP mixture3p006 N⁶-(α-methyl-2-nitrobenzyl)-2′-dATP mixture 3p0635-(α-isopropyl-2-nitrobenzyloxy)methyl-2′-dUTP single 3p0655-(α-isopropyl-2-nitrobenzyloxy)methyl-2′-dCTP single 3p0755-(α-tert-butyl-2-nitrobenzyloxy)methyl-2′-dUTP single 3p0855-(α-tert-butyl-2-nitrobenzyloxy)methyl-2′-dCTP single 4p1355-(α-isopropyl-2-nitrobenzyloxy)methyl-2′-dCTP-linker single 5p085C⁷-(2-nitrobenzyloxy)methyl-2′-dATP 5p098-ds1C⁷-(α-isopropyl-2-nitrobenzyloxy)methyl-2′-dATP (ds1) single 5p098-ds2C⁷-(α-isopropyl-2-nitrobenzyloxy)methyl-2′-dATP (ds2) single 5p107C⁷-(2-nitrobenzyloxy)methyl-2′-dGTP 5p1115-(α-isopropyl-benzyloxy)methyl-2′-dUTP mixture 5p127C⁷-(α-isopropyl-2-nitrobenzyloxy)methyl-2′-dATP-6-FAM single 5p130-LP2C⁷-(α-isopropyl-2-nitrobenzyloxy)methyl-2′-dATP-6-CR110 single 5p143C⁷-(α-isopropyl-2-nitrobenzyloxy)methyl-2′-dGTP mixture 5p143-ds1C⁷-(α-isopropyl-2-nitrobenzyloxy)methyl-2′-dGTP (ds1) single 5p143-ds2C⁷-(α-isopropyl-2-nitrobenzyloxy)methyl-2′-dGTP (ds2) single 5p1455-(benzyloxy)methyl-2′-dUTP 5p147 5-(2-methylbenzyloxy)methyl-2′-dUTP5p149 5-(2-isopropylbenzyloxy)methyl-2′-dUTP 6p0055-(α-isopropyl-2-nitrobenzyloxy)methyl-2′-dUTP-5-R6G single 6p0085-(α-isopropyl-2-nitrobenzyloxy)methyl-2′-dUTP-6-JOE single 6p0105-(2-phenylbenzyloxy)methyl-2′-dUTP 6p0155-(2,6-dimethylbenzyloxy)methyl-2′-dUTP 6p0175-(α-isopropyl-2-nitrobenzyloxy)methyl-2′-dCTP-Cy5 single 6p0245-(2-tert-butylbenzyloxy)methyl-2′-dUTP 6p034C⁷-(α-isopropyl-2-nitrobenzyloxy)methyl-2′-dGTP-6-ROX single 6p036C⁷-(α-isopropyl-2-nitrobenzyloxy)methyl- single 2′-dGTP-dTAMRA-1 6p0445-(α-isopropyl-2-nitrobenzyloxy)methyl-2′-dUTP-6-ROX single 6p057-ds1C⁷-(α-isopropyl-2,6-dinitrobenzyloxy)methyl-2′-dATP (ds1) single6p057-ds2 C⁷-(α-isopropyl-2,6-dinitrobenzyloxy)methyl-2′-dATP (ds2)single 6p063 N⁶-(α-isopropyl-2-nitrobenzyl)-2′-dATP single 6p071C⁷-(α-isopropyl-2-nitrobenzyloxy)methyl- single 2′-dGTP-alexa-fluor-5306p073 C⁷-(α-isopropyl-2-nitrobenzyloxy)methyl-2′-dGTP-6-JOE single6p087-ds1 C⁷-(α-isopropyl-4-methoxy-2-nitrobenzyl- singleoxy)methyl-2′-dATP (ds1) 6p087-ds2C⁷-(α-isopropyl-4-methoxy-2-nitrobenzyl- single oxy)methyl-2′-dATP (ds2)6p094 5-(α-isopropyl-2-nitrobenzyloxy)methyl-2′-dUTP-6-FAM single

Other objects, features and advantages of the present disclosure willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating specific embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.Note that simply because a particular compound is ascribed to oneparticular generic formula doesn't mean that it cannot also belong toanother generic formula.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentdisclosure. The invention may be better understood by reference to oneof these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1. Incorporation Assay: Natural dCTP and a modified dCTP analogwere assayed for incorporation on a template with complementary base“G”.

FIG. 2. X-ray Crystallography data of(S)-1-(2-nitrophenyl)-2-methyl-1-propyl (1S)-camphanate: C₂₀H₂₅NO₆,M=375.41, colorless plate, 0.26×0.24×0.10 mm³, orthorhombic, space groupP2₁2₁2₁ (No. 19), a=11.9268(15), b=11.9812(14), c=13.5488(16) Å,V=1936.1(4) Å³, Z=4, D_(c)=1.288 g/cm³, F₀₀₀=800, MWPC area detector,CuKα radiation, λ=1.54178 Å, T=110(2)K, 2θ_(max)=120.0°, 22896reflections collected, 2665 unique (R_(int)=0.0462). Final GooF=1.009,R1=0.0219, wR2=0.0554, R indices based on 2629 reflections withI>2sigma(I) (refinement on F²), 245 parameters, 0 restraints. Lp andabsorption corrections applied, μ=0.787 mm⁻¹. Absolute structureparameter=0.09(5).

FIG. 3. X-ray crystallography data for(R)-1-(4-iodo-2-nitrophenyl)-2-methyl-1-propyl (1S)-camphanate: Crystaldata for lg₀₁a: C₂₀H₂₄INO₆, M=501.30, colorless plate, 0.30×0.20×0.20mm³, monoclinic, space group P2₁ (No. 4), a=7.5810(15), b=12.446(3),c=11.722(3) Å, β=107.613(10)°, V=1054.2(4) Å³, Z=2, D_(c)=1.579 g/cm³,F₀₀₀=504, CCD area detector, MoKα radiation, λ=0.71073 Å, T=110(2)K,2θ_(max)=50.0°, 24239 reflections collected, 3558 unique(R_(int)=0.0302). Final GooF=1.010, R₁=0.0123, wR2=0.0316, R indicesbased on 3520 reflections with I>2sigma(I) (refinement on F²), 253parameters, 3 restraints. Lp and absorption corrections applied, μ=1.554mm⁻¹. Absolute structure parameter=0.020(9).

FIG. 4. HPLC stack trace of hydrogenolysis of5-benzyloxymethyl-2′-deoxyuridine.

FIG. 5. HPLC stack trace of hydrogenolysis of5-(1-phenyl-2-methyl-propyloxy)methyl-2′-deoxyuridine.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS I. Definitions

When used in the context of a chemical group, “hydrogen” means —H;“hydroxy” means —OH; “oxo” means ═O; “halo” means independently —F, —Cl,—Br or —I; “amino” means —NH₂ (see below for definitions of groupscontaining the term amino, e.g., alkylamino); “hydroxyamino” means—NHOH; “nitro” means —NO₂; imino means ═NH (see below for definitions ofgroups containing the term imino, e.g., alkylimino); “cyano” means —CN;“azido” means —N₃; in a monovalent context “phosphate” means —OP(O)(OH)₂or a deprotonated form thereof; in a divalent context “phosphate” means—OP(O)(OH)O— or a deprotonated form thereof; “mercapto” means —SH;“thio” means ═S; “thioether” means —S—; “sulfonamido” means —NHS(O)₂—(see below for definitions of groups containing the term sulfonamido,e.g., alkylsulfonamido); “sulfonyl” means —S(O)₂— (see below fordefinitions of groups containing the term sulfonyl, e.g.,alkylsulfonyl); “sulfinyl” means —S(O)— (see below for definitions ofgroups containing the term sulfinyl, e.g., alkylsulfinyl); and “silyl”means —SiH₃ (see below for definitions of group(s) containing the termsilyl, e.g., alkylsilyl).

The symbol “—” means a single bond, “═” means a double bond, and “≡”means triple bond. The symbol “

” represents a single bond or a double bond. The symbol “

”, when drawn perpendicularly across a bond indicates a point ofattachment of the group. It is noted that the point of attachment istypically only identified in this manner for larger groups in order toassist the reader in rapidly and unambiguously identifying a point ofattachment. The symbol “

” means a single bond where the group attached to the thick end of thewedge is “out of the page.” The symbol “

” means a single bond where the group attached to the thick end of thewedge is “into the page”. The symbol “

” means a single bond where the conformation is unknown (e.g., either Ror S), the geometry is unknown (e.g., either E or Z) or the compound ispresent as mixture of conformation or geometries (e.g., a 50%/50%mixture).

When a group “R” is depicted as a “floating group” on a ring system, forexample, in the formula:

then R may replace any hydrogen atom attached to any of the ring atoms,including a depicted, implied, or expressly defined hydrogen, so long asa stable structure is formed.

When a group “R” is depicted as a “floating group” on a fused ringsystem, as for example in the formula:

then R may replace any hydrogen attached to any of the ring atoms ofeither of the fuzed rings unless specified otherwise. Replaceablehydrogens include depicted hydrogens (e.g., the hydrogen attached to thenitrogen in the formula above), implied hydrogens (e.g., a hydrogen ofthe formula above that is not shown but understood to be present),expressly defined hydrogens, and optional hydrogens whose presencedepends on the identity of a ring atom (e.g., a hydrogen attached togroup X, when X equals —CH—), so long as a stable structure is formed.In the example depicted, R may reside on either the 5-membered or the6-membered ring of the fused ring system. In the formula above, thesubscript letter “y” immediately following the group “R” enclosed inparentheses, represents a numeric variable. Unless specified otherwise,this variable can be 0, 1, 2, or any integer greater than 2, onlylimited by the maximum number of replaceable hydrogen atoms of the ringor ring system.

When y is 2 and “(R)_(y)” is depicted as a floating group on a ringsystem having one or more ring atoms having two replaceable hydrogens,e.g., a saturated ring carbon, as for example in the formula:

then each of the two R groups can reside on the same or a different ringatom. For example, when R is methyl and both R groups are attached tothe same ring atom, a geminal dimethyl group results. Where specificallyprovided for, two R groups may be taken together to form a divalentgroup, such as one of the divalent groups further defined below. Whensuch a divalent group is attached to the same ring atom, a spirocyclicring structure will result.

When the point of attachment is depicted as “floating”, for example, inthe formula:

then the point of attachment may replace any replaceable hydrogen atomon any of the ring atoms of either of the fuzed rings unless specifiedotherwise.

In the case of a double-bonded R group (e.g., oxo, imino, thio,alkylidene, etc.), any pair of implicit or explicit hydrogen atomsattached to one ring atom can be replaced by the R group. This conceptis exemplified below:

For the groups below, the following parenthetical subscripts furtherdefine the groups as follows: “(Cn)” defines the exact number (n) ofcarbon atoms in the group. “(C≦n)” defines the maximum number (n) ofcarbon atoms that can be in the group, with the minimum number of carbonatoms in such at least one, but otherwise as small as possible for thegroup in question. E.g., it is understood that the minimum number ofcarbon atoms in the group “alkenyl_((C≦8))” is two. For example,“alkoxy_((C≦10))” designates those alkoxy groups having from 1 to 10carbon atoms (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or any rangederivable therein (e.g., 3 to 10 carbon atoms). (Cn-n′) defines both theminimum (n) and maximum number (n′) of carbon atoms in the group.Similarly, “alkyl_((C2-10))” designates those alkyl groups having from 2to 10 carbon atoms (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10, or any rangederivable therein (e.g., 3 to 10 carbon atoms)).

The term “alkyl” when used without the “substituted” modifier refers toa non-aromatic monovalent group with a saturated carbon atom as thepoint of attachment, a linear or branched, cyclo, cyclic or acyclicstructure, no carbon-carbon double or triple bonds, and no atoms otherthan carbon and hydrogen. The groups, —CH₃ (Me), —CH₂CH₃ (Et),—CH₂CH₂CH₃ (n-Pr), —CH(CH₃)₂ (iso-Pr), —CH(CH₂)₂ (cyclopropyl),—CH₂CH₂CH₂CH₃ (n-Bu), —CH(CH₃)CH₂CH₃ (sec-butyl), —CH₂CH(CH₃)₂(iso-butyl), —C(CH₃)₃ (tert-butyl), —CH₂C(CH₃)₃ (neo-pentyl),cyclobutyl, cyclopentyl, cyclohexyl, and cyclohexylmethyl arenon-limiting examples of alkyl groups. The term “substituted alkyl”refers to a non-aromatic monovalent group with a saturated carbon atomas the point of attachment, a linear or branched, cyclo, cyclic oracyclic structure, no carbon-carbon double or triple bonds, and at leastone atom independently selected from the group consisting of N, O, F,Cl, Br, I, Si, P, and S. The following groups are non-limiting examplesof substituted alkyl groups: —CH₂OH, —CH₂Cl, —CH₂Br, —CH₂SH, —CF₃,—CH₂CN, —CH₂C(O)H, —CH₂C(O)OH, —CH₂C(O)OCH₃, —CH₂C(O)NH₂, —CH₂C(O)NHCH₃,—CH₂C(O)CH₃, —CH₂OCH₃, —CH₂OCH₂CF₃, —CH₂OC(O)CH₃, —CH₂NH₂, —CH₂NHCH₃,—CH₂N(CH₃)₂, —CH₂CH₂Cl, —CH₂CH₂OH, —CH₂CF₃, —CH₂CH₂OC(O)CH₃,—CH₂CH₂NHCO₂C(CH₃)₃, and —CH₂Si(CH₃)₃.

The term “alkanediyl” when used without the “substituted” modifierrefers to a non-aromatic divalent group, wherein the alkanediyl group isattached with two σ-bonds, with one or two saturated carbon atom(s) asthe point(s) of attachment, a linear or branched, cyclo, cyclic oracyclic structure, no carbon-carbon double or triple bonds, and no atomsother than carbon and hydrogen. The groups, —CH₂-(methylene), —CH₂CH₂—,—CH₂C(CH₃)₂CH₂—, —CH₂CH₂CH₂—, and

are non-limiting examples of alkanediyl groups. The term “substitutedalkanediyl” refers to a non-aromatic monovalent group, wherein thealkynediyl group is attached with two σ-bonds, with one or two saturatedcarbon atom(s) as the point(s) of attachment, a linear or branched,cyclo, cyclic or acyclic structure, no carbon-carbon double or triplebonds, and at least one atom independently selected from the groupconsisting of N, O, F, Cl, Br, I, Si, P, and S. The following groups arenon-limiting examples of substituted alkanediyl groups: —CH(F)—, —CF₂—,—CH(Cl)—, —CH(OH)—, —CH(OCH₃)—, and —CH₂CH(Cl)—.

The term “alkenyl” when used without the “substituted” modifier refersto a monovalent group with a nonaromatic carbon atom as the point ofattachment, a linear or branched, cyclo, cyclic or acyclic structure, atleast one nonaromatic carbon-carbon double bond, no carbon-carbon triplebonds, and no atoms other than carbon and hydrogen. Non-limitingexamples of alkenyl groups include: —CH═CH₂ (vinyl), —CH═CHCH₃,—CH═CHCH₂CH₃, —CH₂CH═CH₂ (allyl), —CH₂CH═CHCH₃, and —CH═CH—C₆H₅. Theterm “substituted alkenyl” refers to a monovalent group with anonaromatic carbon atom as the point of attachment, at least onenonaromatic carbon-carbon double bond, no carbon-carbon triple bonds, alinear or branched, cyclo, cyclic or acyclic structure, and at least oneatom independently selected from the group consisting of N, O, F, Cl,Br, I, Si, P, and S. The groups, —CH═CHF, —CH═CHCl and —CH═CHBr, arenon-limiting examples of substituted alkenyl groups.

The term “alkenediyl” when used without the “substituted” modifierrefers to a non-aromatic divalent group, wherein the alkenediyl group isattached with two σ-bonds, with two carbon atoms as points ofattachment, a linear or branched, cyclo, cyclic or acyclic structure, atleast one nonaromatic carbon-carbon double bond, no carbon-carbon triplebonds, and no atoms other than carbon and hydrogen. The groups, —CH═CH—,—CH═C(CH₃)CH₂—, —CH═CHCH₂—, and

are non-limiting examples of alkenediyl groups. The term “substitutedalkenediyl” refers to a non-aromatic divalent group, wherein thealkenediyl group is attached with two σ-bonds, with two carbon atoms aspoints of attachment, a linear or branched, cyclo, cyclic or acyclicstructure, at least one nonaromatic carbon-carbon double bond, nocarbon-carbon triple bonds, and at least one atom independently selectedfrom the group consisting of N, O, F, Cl, Br, I, Si, P, and S. Thefollowing groups are non-limiting examples of substituted alkenediylgroups: —CF═CH—, —C(OH)═CH—, and —CH₂CH═C(Cl)—.

The term “alkynyl” when used without the “substituted” modifier refersto a monovalent group with a nonaromatic carbon atom as the point ofattachment, a linear or branched, cyclo, cyclic or acyclic structure, atleast one carbon-carbon triple bond, and no atoms other than carbon andhydrogen. The groups, —C≡CH, —C≡CCH₃, —C≡CC₆H₅ and —CH₂C≡CCH₃, arenon-limiting examples of alkynyl groups. The term “substituted alkynyl”refers to a monovalent group with a nonaromatic carbon atom as the pointof attachment and at least one carbon-carbon triple bond, a linear orbranched, cyclo, cyclic or acyclic structure, and at least one atomindependently selected from the group consisting of N, O, F, Cl, Br, I,Si, P, and S. The group, —C≡CSi(CH₃)₃, is a non-limiting example of asubstituted alkynyl group.

The term “alkynediyl” when used without the “substituted” modifierrefers to a non-aromatic divalent group, wherein the alkynediyl group isattached with two σ-bonds, with two carbon atoms as points ofattachment, a linear or branched, cyclo, cyclic or acyclic structure, atleast one carbon-carbon triple bond, and no atoms other than carbon andhydrogen. The groups, —C≡C—, —C≡CCH₂—, and —C≡CCH(CH₃)— are non-limitingexamples of alkynediyl groups. The term “substituted alkynediyl” refersto a non-aromatic divalent group, wherein the alkynediyl group isattached with two σ-bonds, with two carbon atoms as points ofattachment, a linear or branched, cyclo, cyclic or acyclic structure, atleast one carbon-carbon triple bond, and at least one atom independentlyselected from the group consisting of N, O, F, Cl, Br, I, Si, P, and S.The groups —C≡CCFH— and —C≡CHCH(Cl)— are non-limiting examples ofsubstituted alkynediyl groups.

The term “aryl” when used without the “substituted” modifier refers to amonovalent group with an aromatic carbon atom as the point ofattachment, said carbon atom forming part of one or more six-memberedaromatic ring structure(s) wherein the ring atoms are all carbon, andwherein the monovalent group consists of no atoms other than carbon andhydrogen. Non-limiting examples of aryl groups include phenyl (Ph),methylphenyl, (dimethyl)phenyl, —C₆H₄—CH₂CH₃ (ethylphenyl),—C₆H₄—CH₂CH₂CH₃ (propylphenyl), —C₆H₄CH(CH₃)₂, —C₆H₄CH(CH₂)₂,—C₆H₃(CH₃)CH₂CH₃ (methylethylphenyl), —C₆H₄CH═CH₂ (vinylphenyl),—C₆H₄CH═CHCH₃, —C₆H₄C≡CH, —C₆H₄C≡CCH₃, naphthyl, and the monovalentgroup derived from biphenyl. The term “substituted aryl” refers to amonovalent group with an aromatic carbon atom as the point ofattachment, said carbon atom forming part of one or more six-memberedaromatic ring structure(s) wherein the ring atoms are all carbon, andwherein the monovalent group further has at least one atom independentlyselected from the group consisting of N, O, F, Cl, Br, I, Si, P, and S.Non-limiting examples of substituted aryl groups include the groups:—C₆H₄F, —C₆H₄Cl, —C₆H₄Br, —C₆H₄I, —C₆H₄OH, —C₆H₄OCH₃, —C₆H₄OCH₂CH₃,—C₆H₄OC(O)CH₃, —C₆H₄NH₂, —C₆H₄NHCH₃, —C₆H₄N(CH₃)₂, —C₆H₄CH₂OH,—C₆H₄CH₂OC(O)CH₃, —C₆H₄CH₂NH₂, —C₆H₄CF₃, —C₆H₄CN, —C₆H₄CHO, —C₆H₄CHO,—C₆H₄C(O)CH₃, —C₆H₄C(O)C₆H₅, —C₆H₄CO₂H, —C₆H₄CO₂CH₃, —C₆H₄CONH₂,—C₆H₄CONHCH₃, and —C₆H₄CON(CH₃)₂.

The term “arenediyl” when used without the “substituted” modifier refersto a divalent group, wherein the arenediyl group is attached with twoσ-bonds, with two aromatic carbon atoms as points of attachment, saidcarbon atoms forming part of one or more six-membered aromatic ringstructure(s) wherein the ring atoms are all carbon, and wherein themonovalent group consists of no atoms other than carbon and hydrogen.Non-limiting examples of arenediyl groups include:

The term “substituted arenediyl” refers to a divalent group, wherein thearenediyl group is attached with two σ-bonds, with two aromatic carbonatoms as points of attachment, said carbon atoms forming part of one ormore six-membered aromatic rings structure(s), wherein the ring atomsare carbon, and wherein the divalent group further has at least one atomindependently selected from the group consisting of N, O, F, Cl, Br, I,Si, P, and S.

The term “aralkyl” when used without the “substituted” modifier refersto the monovalent group -alkanediyl-aryl, in which the terms alkanediyland aryl are each used in a manner consistent with the definitionsprovided above. Non-limiting examples of aralkyls are: phenylmethyl(benzyl, Bn), 1-phenyl-ethyl, 2-phenyl-ethyl, indenyl and2,3-dihydro-indenyl, provided that indenyl and 2,3-dihydro-indenyl areonly examples of aralkyl in so far as the point of attachment in eachcase is one of the saturated carbon atoms. When the term “aralkyl” isused with the “substituted” modifier, either one or both the alkanediyland the aryl is substituted. Non-limiting examples of substitutedaralkyls are: (3-chlorophenyl)-methyl, 2-oxo-2-phenyl-ethyl(phenylcarbonylmethyl), 2-chloro-2-phenyl-ethyl, chromanyl where thepoint of attachment is one of the saturated carbon atoms, andtetrahydroquinolinyl where the point of attachment is one of thesaturated atoms.

The term “heteroaryl” when used without the “substituted” modifierrefers to a monovalent group with an aromatic carbon atom or nitrogenatom as the point of attachment, said carbon atom or nitrogen atomforming part of an aromatic ring structure wherein at least one of thering atoms is nitrogen, oxygen or sulfur, and wherein the monovalentgroup consists of no atoms other than carbon, hydrogen, aromaticnitrogen, aromatic oxygen and aromatic sulfur. Non-limiting examples ofaryl groups include acridinyl, furanyl, imidazoimidazolyl,imidazopyrazolyl, imidazopyridinyl, imidazopyrimidinyl, indolyl,indazolinyl, methylpyridyl, oxazolyl, phenylimidazolyl, pyridyl,pyrrolyl, pyrimidyl, pyrazinyl, quinolyl, quinazolyl, quinoxalinyl,tetrahydroquinolinyl, thienyl, triazinyl, pyrrolopyridinyl,pyrrolopyrimidinyl, pyrrolopyrazinyl, pyrrolotriazinyl,pyrroloimidazolyl, chromenyl (where the point of attachment is one ofthe aromatic atoms), and chromanyl (where the point of attachment is oneof the aromatic atoms). The term “substituted heteroaryl” refers to amonovalent group with an aromatic carbon atom or nitrogen atom as thepoint of attachment, said carbon atom or nitrogen atom forming part ofan aromatic ring structure wherein at least one of the ring atoms isnitrogen, oxygen or sulfur, and wherein the monovalent group further hasat least one atom independently selected from the group consisting ofnon-aromatic nitrogen, non-aromatic oxygen, non aromatic sulfur F, Cl,Br, I, Si, and P.

The term “heteroarenediyl” when used without the “substituted” modifierrefers to a divalent group, wherein the heteroarenediyl group isattached with two σ-bonds, with an aromatic carbon atom or nitrogen atomas the point of attachment, said carbon atom or nitrogen atom formingpart of one or more aromatic ring structure(s) wherein at least one ofthe ring atoms is nitrogen, oxygen or sulfur, and wherein the divalentgroup consists of no atoms other than carbon, hydrogen, aromaticnitrogen, aromatic oxygen and aromatic sulfur. Non-limiting examples ofheteroarenediyl groups include:

The term “substituted heteroarenediyl” refers to a divalent group,wherein the heteroarenediyl group is attached with two σ-bonds, with anaromatic carbon atom or nitrogen atom as points of attachment, saidcarbon atom or nitrogen atom forming part of one or more six-memberedaromatic ring structure(s), wherein at least one of the ring atoms isnitrogen, oxygen or sulfur, and wherein the divalent group further hasat least one atom independently selected from the group consisting ofnon-aromatic nitrogen, non-aromatic oxygen, non aromatic sulfur F, Cl,Br, I, Si, and P.

The term “heteroaralkyl” when used without the “substituted” modifierrefers to the monovalent group -alkanediyl-heteroaryl, in which theterms alkanediyl and heteroaryl are each used in a manner consistentwith the definitions provided above. Non-limiting examples of aralkylsare: pyridylmethyl, and thienylmethyl. When the term “heteroaralkyl” isused with the “substituted” modifier, either one or both the alkanediyland the heteroaryl is substituted.

The term “acyl” when used without the “substituted” modifier refers to amonovalent group with a carbon atom of a carbonyl group as the point ofattachment, further having a linear or branched, cyclo, cyclic oracyclic structure, further having no additional atoms that are notcarbon or hydrogen, beyond the oxygen atom of the carbonyl group. Thegroups, —CHO, —C(O)CH₃ (acetyl, Ac), —C(O)CH₂CH₃, —C(O)CH₂CH₂CH₃,—C(O)CH(CH₃)₂, —C(O)CH(CH₂)₂, —C(O)C₆H₅, —C(O)C₆H₄CH₃, —C(O)C₆H₄CH₂CH₃,—COC₆H₃(CH₃)₂, and —C(O)CH₂C₆H₅, are non-limiting examples of acylgroups. The term “acyl” therefore encompasses, but is not limited togroups sometimes referred to as “alkyl carbonyl” and “aryl carbonyl”groups. The term “substituted acyl” refers to a monovalent group with acarbon atom of a carbonyl group as the point of attachment, furtherhaving a linear or branched, cyclo, cyclic or acyclic structure, furtherhaving at least one atom, in addition to the oxygen of the carbonylgroup, independently selected from the group consisting of N, O, F, Cl,Br, I, Si, P, and S. The groups, —C(O)CH₂CF₃, —CO₂H (carboxyl), —CO₂CH₃(methylcarboxyl), —CO₂CH₂CH₃, —CO₂CH₂CH₂CH₃, —CO₂C₆H₅, —CO₂CH(CH₃)₂,—CO₂CH(CH₂)₂, —C(O)NH₂ (carbamoyl), —C(O)NHCH₃, —C(O)NHCH₂CH₃,—CONHCH(CH₃)₂, —CONHCH(CH₂)₂, —CON(CH₃)₂, —CONHCH₂CF₃, —CO-pyridyl,—CO-imidazoyl, and —C(O)N₃, are non-limiting examples of substitutedacyl groups. The term “substituted acyl” encompasses, but is not limitedto, “heteroaryl carbonyl” groups.

The term “alkylidene” when used without the “substituted” modifierrefers to the divalent group ═CRR′, wherein the alkylidene group isattached with one σ-bond and one π-bond, in which R and R′ areindependently hydrogen, alkyl, or R and R′ are taken together torepresent alkanediyl. Non-limiting examples of alkylidene groupsinclude: ═CH₂, ═CH(CH₂CH₃), and ═C(CH₃)₂. The term “substitutedalkylidene” refers to the group ═CRR′, wherein the alkylidene group isattached with one σ-bond and one π-bond, in which R and R′ areindependently hydrogen, alkyl, substituted alkyl, or R and R′ are takentogether to represent a substituted alkanediyl, provided that either oneof R and R′ is a substituted alkyl or R and R′ are taken together torepresent a substituted alkanediyl.

The term “alkoxy” when used without the “substituted” modifier refers tothe group —OR, in which R is an alkyl, as that term is defined above.Non-limiting examples of alkoxy groups include: —OCH₃, —OCH₂CH₃,—OCH₂CH₂CH₃, —OCH(CH₃)₂, —OCH(CH₂)₂, —O-cyclopentyl, and —O-cyclohexyl.The term “substituted alkoxy” refers to the group —OR, in which R is asubstituted alkyl, as that term is defined above. For example, —OCH₂CF₃is a substituted alkoxy group.

Similarly, the terms “alkenyloxy”, “alkynyloxy”, “aryloxy”, “aralkoxy”,“heteroaryloxy”, “heteroaralkoxy” and “acyloxy”, when used without the“substituted” modifier, refers to groups, defined as —OR, in which R isalkenyl, alkynyl, aryl, aralkyl, heteroaryl, heteroaralkyl and acyl,respectively, as those terms are defined above. When any of the termsalkenyloxy, alkynyloxy, aryloxy, aralkyloxy and acyloxy is modified by“substituted,” it refers to the group —OR, in which R is substitutedalkenyl, alkynyl, aryl, aralkyl, heteroaryl, heteroaralkyl and acyl,respectively.

The term “alkylamino” when used without the “substituted” modifierrefers to the group —NHR, in which R is an alkyl, as that term isdefined above. Non-limiting examples of alkylamino groups include:—NHCH₃, —NHCH₂CH₃, —NHCH₂CH₂CH₃, —NHCH(CH₃)₂, —NHCH(CH₂)₂,—NHCH₂CH₂CH₂CH₃, —NHCH(CH₃)CH₂CH₃, —NHCH₂CH(CH₃)₂, —NHC(CH₃)₃,—NH-cyclopentyl, and —NH-cyclohexyl. The term “substituted alkylamino”refers to the group —NHR, in which R is a substituted alkyl, as thatterm is defined above. For example, —NHCH₂CF₃ is a substitutedalkylamino group.

The term “dialkylamino” when used without the “substituted” modifierrefers to the group —NRR′, in which R and R′ can be the same ordifferent alkyl groups, or R and R′ can be taken together to representan alkanediyl having two or more saturated carbon atoms, at least two ofwhich are attached to the nitrogen atom. Non-limiting examples ofdialkylamino groups include: —NHC(CH₃)₃, —N(CH₃)CH₂CH₃, —N(CH₂CH₃)₂,N-pyrrolidinyl, and N-piperidinyl. The term “substituted dialkylamino”refers to the group —NRR′, in which R and R′ can be the same ordifferent substituted alkyl groups, one of R or R′ is an alkyl and theother is a substituted alkyl, or R and R′ can be taken together torepresent a substituted alkanediyl with two or more saturated carbonatoms, at least two of which are attached to the nitrogen atom.

The terms “alkoxyamino”, “alkenylamino”, “alkynylamino”, “arylamino”,“aralkylamino”, “heteroarylamino”, “heteroaralkylamino”, and“alkylsulfonylamino” when used without the “substituted” modifier,refers to groups, defined as —NHR, in which R is alkoxy, alkenyl,alkynyl, aryl, aralkyl, heteroaryl, heteroaralkyl and alkylsulfonyl,respectively, as those terms are defined above. A non-limiting exampleof an arylamino group is —NHC₆H₅. When any of the terms alkoxyamino,alkenylamino, alkynylamino, arylamino, aralkylamino, heteroarylamino,heteroaralkylamino and alkylsulfonylamino is modified by “substituted,”it refers to the group —NHR, in which R is substituted alkoxy, alkenyl,alkynyl, aryl, aralkyl, heteroaryl, heteroaralkyl and alkylsulfonyl,respectively.

The term “amido” (acylamino), when used without the “substituted”modifier, refers to the group —NHR, in which R is acyl, as that term isdefined above. A non-limiting example of an acylamino group is—NHC(O)CH₃. When the term amido is used with the “substituted” modifier,it refers to groups, defined as —NHR, in which R is substituted acyl, asthat term is defined above. The groups —NHC(O)OCH₃ and —NHC(O)NHCH₃ arenon-limiting examples of substituted amido groups.

The term “alkylimino” when used without the “substituted” modifierrefers to the group ═NR, wherein the alkylimino group is attached withone σ-bond and one π-bond, in which R is an alkyl, as that term isdefined above. Non-limiting examples of alkylimino groups include:═NCH₃, ═NCH₂CH₃ and ═N-cyclohexyl. The term “substituted alkylimino”refers to the group ═NR, wherein the alkylimino group is attached withone σ-bond and one π-bond, in which R is a substituted alkyl, as thatterm is defined above. For example, ═NCH₂CF₃ is a substituted alkyliminogroup.

Similarly, the terms “alkenylimino”, “alkynylimino”, “arylimino”,“aralkylimino”, “heteroarylimino”, “heteroaralkylimino” and “acylimino”,when used without the “substituted” modifier, refers to groups, definedas ═NR, wherein the alkylimino group is attached with one σ-bond and oneπ-bond, in which R is alkenyl, alkynyl, aryl, aralkyl, heteroaryl,heteroaralkyl and acyl, respectively, as those terms are defined above.When any of the terms alkenylimino, alkynylimino, arylimino,aralkylimino and acylimino is modified by “substituted,” it refers tothe group ═NR, wherein the alkylimino group is attached with one σ-bondand one π-bond, in which R is substituted alkenyl, alkynyl, aryl,aralkyl, heteroaryl, heteroaralkyl and acyl, respectively.

The term “fluoroalkyl” when used without the “substituted” modifierrefers to an alkyl, as that term is defined above, in which one or morefluorines have been substituted for hydrogens. The groups, —CH₂F, —CF₂H,—CF₃, and —CH₂CF₃ are non-limiting examples of fluoroalkyl groups. Theterm “substituted fluoroalkyl” refers to a non-aromatic monovalent groupwith a saturated carbon atom as the point of attachment, a linear orbranched, cyclo, cyclic or acyclic structure, at least one fluorineatom, no carbon-carbon double or triple bonds, and at least one atomindependently selected from the group consisting of N, O, Cl, Br, I, Si,P, and S. The following group is a non-limiting example of a substitutedfluoroalkyl: —CFHOH.

The term “alkylphosphate” when used without the “substituted” modifierrefers to the group —OP(O)(OH)(OR), in which R is an alkyl, as that termis defined above. Non-limiting examples of alkylphosphate groupsinclude: —OP(O)(OH)(OMe) and —OP(O)(OH)(OEt). The term “substitutedalkylphosphate” refers to the group —OP(O)(OH)(OR), in which R is asubstituted alkyl, as that term is defined above.

The term “dialkylphosphate” when used without the “substituted” modifierrefers to the group —OP(O)(OR)(OR′), in which R and R′ can be the sameor different alkyl groups, or R and R′ can be taken together torepresent an alkanediyl having two or more saturated carbon atoms, atleast two of which are attached via the oxygen atoms to the phosphorusatom. Non-limiting examples of dialkylphosphate groups include:—OP(O)(OMe)₂, —OP(O)(OEt)(OMe) and —OP(O)(OEt)₂. The term “substituteddialkylphosphate” refers to the group —OP(O)(OR)(OR′), in which R and R′can be the same or different substituted alkyl groups, one of R or R′ isan alkyl and the other is a substituted alkyl, or R and R′ can be takentogether to represent a substituted alkanediyl with two or moresaturated carbon atoms, at least two of which are attached via theoxygen atoms to the phosphorous.

The term “alkylthio” when used without the “substituted” modifier refersto the group —SR, in which R is an alkyl, as that term is defined above.Non-limiting examples of alkylthio groups include: —SCH₃, —SCH₂CH₃,—SCH₂CH₂CH₃, —SCH(CH₃)₂, —SCH(CH₂)₂, —S-cyclopentyl, and —S-cyclohexyl.The term “substituted alkylthio” refers to the group —SR, in which R isa substituted alkyl, as that term is defined above. For example,—SCH₂CF₃ is a substituted alkylthio group.

Similarly, the terms “alkenylthio”, “alkynylthio”, “arylthio”,“aralkylthio”, “heteroarylthio”, “heteroaralkylthio”, and “acylthio”,when used without the “substituted” modifier, refers to groups, definedas —SR, in which R is alkenyl, alkynyl, aryl, aralkyl, heteroaryl,heteroaralkyl and acyl, respectively, as those terms are defined above.When any of the terms alkenylthio, alkynylthio, arylthio, aralkylthio,heteroarylthio, heteroaralkylthio, and acylthio is modified by“substituted,” it refers to the group —SR, in which R is substitutedalkenyl, alkynyl, aryl, aralkyl, heteroaryl, heteroaralkyl and acyl,respectively.

The term “thioacyl” when used without the “substituted” modifier refersto a monovalent group with a carbon atom of a thiocarbonyl group as thepoint of attachment, further having a linear or branched, cyclo, cyclicor acyclic structure, further having no additional atoms that are notcarbon or hydrogen, beyond the sulfur atom of the carbonyl group. Thegroups, —CHS, —C(S)CH₃, —C(S)CH₂CH₃, —C(S)CH₂CH₂CH₃, —C(S)CH(CH₃)₂,—C(S)CH(CH₂)₂, —C(S)C₆H₅, —C(S)C₆H₄CH₃, —C(S)C₆H₄CH₂CH₃,—C(S)C₆H₃(CH₃)₂, and —C(S)CH₂C₆H₅, are non-limiting examples of thioacylgroups. The term “thioacyl” therefore encompasses, but is not limitedto, groups sometimes referred to as “alkyl thiocarbonyl” and “arylthiocarbonyl” groups. The term “substituted thioacyl” refers to aradical with a carbon atom as the point of attachment, the carbon atombeing part of a thiocarbonyl group, further having a linear or branched,cyclo, cyclic or acyclic structure, further having at least one atom, inaddition to the sulfur atom of the carbonyl group, independentlyselected from the group consisting of N, O, F, Cl, Br, I, Si, P, and S.The groups, —C(S)CH₂CF₃, —C(S)O₂H, —C(S)OCH₃, —C(S)OCH₂CH₃,—C(S)OCH₂CH₂CH₃, —C(S)OC₆H₅, —C(S)OCH(CH₃)₂, —C(S)OCH(CH₂)₂, —C(S)NH₂,and —C(S)NHCH₃, are non-limiting examples of substituted thioacylgroups. The term “substituted thioacyl” encompasses, but is not limitedto, “heteroaryl thiocarbonyl” groups.

The term “alkylsulfonyl” when used without the “substituted” modifierrefers to the group —S(O)₂R, in which R is an alkyl, as that term isdefined above. Non-limiting examples of alkylsulfonyl groups include:—S(O)₂CH₃, —S(O)₂CH₂CH₃, —S(O)₂CH₂CH₂CH₃, —S(O)₂CH(CH₃)₂,—S(O)₂CH(CH₂)₂, —S(O)₂-cyclopentyl, and —S(O)₂-cyclohexyl. The term“substituted alkylsulfonyl” refers to the group —S(O)₂R, in which R is asubstituted alkyl, as that term is defined above. For example,—S(O)₂CH₂CF₃ is a substituted alkylsulfonyl group.

Similarly, the terms “alkenylsulfonyl”, “alkynylsulfonyl”,“arylsulfonyl”, “aralkylsulfonyl”, “heteroarylsulfonyl”, and“heteroaralkylsulfonyl” when used without the “substituted” modifier,refers to groups, defined as —S(O)₂R, in which R is alkenyl, alkynyl,aryl, aralkyl, heteroaryl, and heteroaralkyl, respectively, as thoseterms are defined above. When any of the terms alkenylsulfonyl,alkynylsulfonyl, arylsulfonyl, aralkylsulfonyl, heteroarylsulfonyl, andheteroaralkylsulfonyl is modified by “substituted,” it refers to thegroup —S(O)₂R, in which R is substituted alkenyl, alkynyl, aryl,aralkyl, heteroaryl and heteroaralkyl, respectively.

The term “alkylsulfinyl” when used without the “substituted” modifierrefers to the group —S(O)R, in which R is an alkyl, as that term isdefined above. Non-limiting examples of alkylsulfinyl groups include:—S(O)CH₃, —S(O)CH₂CH₃, —S(O)CH₂CH₂CH₃, —S(O)CH(CH₃)₂, —S(O)CH(CH₂)₂,—S(O)-cyclopentyl, and —S(O)-cyclohexyl. The term “substitutedalkylsulfinyl” refers to the group —S(O)R, in which R is a substitutedalkyl, as that term is defined above. For example, —S(O)CH₂CF₃ is asubstituted alkylsulfinyl group.

Similarly, the terms “alkenylsulfinyl”, “alkynylsulfinyl”,“arylsulfinyl”, “aralkylsulfinyl”, “heteroarylsulfinyl”, and“heteroaralkylsulfinyl” when used without the “substituted” modifier,refers to groups, defined as —S(O)R, in which R is alkenyl, alkynyl,aryl, aralkyl, heteroaryl, and heteroaralkyl, respectively, as thoseterms are defined above. When any of the terms alkenylsulfinyl,alkynylsulfinyl, arylsulfinyl, aralkylsulfinyl, heteroarylsulfinyl, andheteroaralkylsulfinyl is modified by “substituted,” it refers to thegroup —S(O)R, in which R is substituted alkenyl, alkynyl, aryl, aralkyl,heteroaryl and heteroaralkyl, respectively.

The term “alkylammonium” when used without the “substituted” modifierrefers to a group, defined as —NH₂R⁺, —NHRR′⁺, or —NRR′R″⁺, in which R,R′ and R″ are the same or different alkyl groups, or any combination oftwo of R, R′ and R″ can be taken together to represent an alkanediyl.Non-limiting examples of alkylammonium cation groups include:—NH₂(CH₃)⁺, —NH₂(CH₂CH₃)⁺, —NH₂(CH₂CH₂CH₃)⁺, —NH(CH₃)₂ ⁺, —NH(CH₂CH₃)₂⁺, —NH(CH₂CH₂CH₃)₂ ⁺, —N(CH₃)₃ ⁺, —N(CH₃)(CH₂CH₃)₂ ⁺, —N(CH₃)₂(CH₂CH₃)⁺,—NH₂C(CH₃)₃ ⁺, —NH(cyclopentyl)₂ ⁺, and —NH₂(cyclohexyl)⁺. The term“substituted alkylammonium” refers —NH₂R⁺, —NHRR′⁺, or —NRR′R″⁺, inwhich at least one of R, R′ and R″ is a substituted alkyl or two of R,R′ and R″ can be taken together to represent a substituted alkanediyl.When more than one of R, R′ and R″ is a substituted alkyl, they can bethe same of different. Any of R, R′ and R″ that are not eithersubstituted alkyl or substituted alkanediyl, can be either alkyl, eitherthe same or different, or can be taken together to represent aalkanediyl with two or more carbon atoms, at least two of which areattached to the nitrogen atom shown in the formula.

The term “alkylsulfonium” when used without the “substituted” modifierrefers to the group —SRR′⁺, in which R and R′ can be the same ordifferent alkyl groups, or R and R′ can be taken together to representan alkanediyl. Non-limiting examples of alkylsulfonium groups include:—SH(CH₃)⁺, —SH(CH₂CH₃)⁺, —SH(CH₂CH₂CH₃)⁺, —S(CH₃)₂ ⁺, —S(CH₂CH₃)₂ ⁺,—S(CH₂CH₂CH₃)₂ ⁺, —SH(cyclopentyl)⁺, and —SH(cyclohexyl)⁺. The term“substituted alkylsulfonium” refers to the group —SRR′⁺, in which R andR′ can be the same or different substituted alkyl groups, one of R or R′is an alkyl and the other is a substituted alkyl, or R and R′ can betaken together to represent a substituted alkanediyl. For example,—SH(CH₂CF₃)⁺ is a substituted alkylsulfonium group.

The term “alkylsilyl” when used without the “substituted” modifierrefers to a monovalent group, defined as —SiH₂R, —SiHRR′, or —SiRR′R″,in which R, R′ and R″ can be the same or different alkyl groups, or anycombination of two of R, R′ and R″ can be taken together to represent analkanediyl. The groups, —SiH₂CH₃, —SiH(CH₃)₂, —Si(CH₃)₃ and—Si(CH₃)₂C(CH₃)₃, are non-limiting examples of unsubstituted alkylsilylgroups. The term “substituted alkylsilyl” refers —SiH₂R, —SiHRR′, or—SiRR′R″, in which at least one of R, R′ and R″ is a substituted alkylor two of R, R′ and R″ can be taken together to represent a substitutedalkanediyl. When more than one of R, R′ and R″ is a substituted alkyl,they can be the same of different. Any of R, R′ and R″ that are noteither substituted alkyl or substituted alkanediyl, can be either alkyl,either the same or different, or can be taken together to represent aalkanediyl with two or more saturated carbon atoms, at least two ofwhich are attached to the silicon atom.

In addition, atoms making up the compounds of the present invention areintended to include all isotopic forms of such atoms. Isotopes, as usedherein, include those atoms having the same atomic number but differentmass numbers. By way of general example and without limitation, isotopesof hydrogen include tritium and deuterium, and isotopes of carboninclude ¹³C and ¹⁴C. Similarly, it is contemplated that one or morecarbon atom(s) of a compound of the present invention may be replaced bya silicon atom(s). Furthermore, it is contemplated that one or moreoxygen atom(s) of a compound of the present invention may be replaced bya sulfur or selenium atom(s).

A compound having a formula that is represented with a dashed bond isintended to include the formulae optionally having zero, one or moredouble bonds. Thus, for example, the structure

includes the structures

As will be understood by a person of skill in the art, no one such ringatom forms part of more than one double bond.

Any undefined valency on an atom of a structure shown in thisapplication implicitly represents a hydrogen atom bonded to the atom.

As used herein, a “chiral auxiliary” refers to a removable chiral groupthat is capable of influencing the stereoselectivity of a reaction.Persons of skill in the art are familiar with such compounds, and manyare commercially available.

The terms “nucleotide base”, “nucleobase” or simply “base”, as usedherein, refers to a substituted or unsubstituted nitrogen-containingparent heteroaromatic ring of a type that is commonly found in nucleicacids, as well as natural, substituted, modified, or engineered variantsor analogs of the same. In a typical embodiment, the nucleobase iscapable of forming Watson-Crick and/or Hoogsteen hydrogen bonds with anappropriately complementary nucleobase. Exemplary nucleobases include,but are not limited to,

-   -   purines such as 2-aminopurine, 2,6-diaminopurine, adenine (A),        ethenoadenine, N⁶-Δ²-isopentenyladenine (6iA),        N⁶-Δ²-isopentenyl-2-methylthioadenine (2 ms6iA),        N⁶-methyladenine, guanine (G), isoguanine, N²-dimethylguanine        (dmG), 7-methylguanine (7mG), 2-thiopyrimidine, 6-thioguanine        (6sG), hypoxanthine and O⁶-methylguanine;    -   7-deaza-purines such as 7-deazaadenine (7-deaza-A),        7-deazaguanine (7-deaza-G), 7-deaza-7-hydroxymethyladenine,        7-deaza-7-aminomethyladenine and 7-deaza-7-hydroxymethylguanine;    -   pyrimidines such as cytosine (C), 5-propynylcytosine,        isocytosine, 5-hydroxylmethylcytosine (HOMeC),        5-aminomethyl-cytosine, thymine (T), 4-thiothymine (4sT),        5,6-dihydrothymine, O⁴-methylthymine, uracil (U), 4-thiouracil        (4sU), 5-hydroxylmethyluracil (HOMeU), 5-aminomethyl-uracil, and        5,6-dihydrouracil (dihydrouracil; D);    -   indoles such as nitroindole and 4-methylindole; pyrroles such as        nitropyrrole; nebularine; base (Y); etc.        Additional exemplary nucleobases can be found in Lehninger,        2005, which is incorporated by reference, and the references        cited therein.

The term “nucleoside” as used herein, refers to a glycosylamineconsisting of a nucleobase bound to a five-carbon sugar, typically aribose or a deoxyribose. Examples of these include, but are not limitedto, cytidine, 2′-deoxycytidine, 5-hydroxylmethylcytidine,2′-deoxy-5-hydroxylmethylcytidine, 5-aminomethylcytidine,2′-deoxy-5-aminomethylcytidine, uridine, 2′-deoxyuridine,5-hydroxylmethyluridine, 2′-deoxy-5-hydroxylmethyluridine,5-aminomethyluridine, 2′-deoxy-5-aminomethyluridine, adenosine,2′-deoxyadenosine, 7-deaza-7-hydroxymethyladenosine,2′-deoxy-7-deaza-7-hydroxymethyladenosine,7-deaza-7-aminomethyladenosine,2′-deoxy-7-deaza-7-amino-methyladenosine, guanosine, 2′-deoxyguanosine,7-deaza-7-hydroxymethyl guanosine, 2′-deoxy-7-deaza-7-hydroxymethyl,7-deaza-7-aminomethyl guanosine, 2′-deoxy-7-deaza-7-aminomethylguanosine, thymidine, and 2′-deoxythymidine.

A “nucleotide” is composed of a nucleoside with one, two, three or morephosphate groups bound in a chain to the 5-carbon sugar of thenucleoside.

Unless specified otherwise, a “linker” refers to one or more divalentgroups (linking members) that function as a covalently-bonded molecularbridge between two other groups. A linker may contain one or morelinking members and one or more types of linking members. Exemplarylinking members include: —C(O)NH—, —C(O)O—, —NH—, —S—, —S(O)_(n)— wheren is 0, 1 or 2, —O—, —OP(O)(OH)O—, —OP(O)(O⁻)O—, alkanediyl, alkenediyl,alkynediyl, arenediyl, heteroarenediyl, or combinations thereof. Somelinkers have pendant side chains or pendant functional groups (or both).Examples of such pendant moieties are hydrophilicity modifiers, forexample, solubilizing groups like, e.g., —SO₃H or —SO₃ ⁻. In someembodiments, a linker may connect a reporter to another moiety such as achemically, photochemically or enzymatically reactive group (e.g., acleavable or non-cleavable terminating moiety). In other embodiments, alinker connects a reporter to a biological and non-biological component,for example, a nucleobase, a nucleoside or a nucleotide. In furtherembodiments, a linker connects chemically reactive groups to anucleobase, a nucleoside or a nucleotide. The moiety formed by a linkerbonded to a reporter may be designated -L-Reporter. Depending on suchfactors as the molecules to be linked and the conditions in which themethod of strand synthesis is performed, the linker may vary in lengthand composition for optimizing properties such as stability, length,FRET efficiency, resistance to certain chemicals and/or temperatureparameters, and be of sufficient stereo-selectivity or size to operablylink a label to a nucleotide such that the resultant conjugate is usefulin optimizing a polymerization reaction. Linkers can be employed usingstandard chemical techniques and include but not limited to, aminelinkers for attaching labels to nucleotides (see, for example, Hobbs andTrainor, U.S. Pat. No. 5,151,507, which is incorporated herein byreference); a linker typically contain a primary or secondary amine foroperably linking a label to a nucleotide; and a rigid hydrocarbon armadded to a nucleotide base (see, for example, Service, 1998, which isincorporated herein by reference). Some exemplary linking methodologiesfor attachment of reporters to base molecules are provided in U.S. Pat.Nos. 4,439,356 and 5,188,934; European Patent Application 87310256.0;International Application PCT/US90/05565 and Barone et al., 2001, eachof which is incorporated herein by reference in its entirety.

A “cleavable linker” is a linker that has one or more cleavable groupsthat may be broken by the result of a reaction or condition. The term“cleavable group” refers to a moiety that allows for release of aportion, e.g., a fluorogenic or fluorescent moiety. Such cleavage istypically chemically, photochemically or enzymatically mediated.Exemplary enzymatically cleavable groups include phosphates, or peptidesequences.

The use of the word “a” or “an,” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.”

Throughout this application, the term “about” is used to indicate that avalue includes the inherent variation of error for the device, themethod being employed to determine the value, or the variation thatexists among the study subjects.

The terms “comprise,” “have” and “include” are open-ended linking verbs.Any forms or tenses of one or more of these verbs, such as “comprises,”“comprising,” “has,” “having,” “includes” and “including,” are alsoopen-ended. For example, any method that “comprises,” “has” or“includes” one or more steps is not limited to possessing only those oneor more steps and also covers other unlisted steps.

The term “effective,” as that term is used in the specification and/orclaims, means adequate to accomplish a desired, expected, or intendedresult.

The term “hydrate” when used as a modifier to a compound means that thecompound has less than one (e.g., hemihydrate), one (e.g., monohydrate),or more than one (e.g., dihydrate) water molecules associated with eachcompound molecule, such as in solid forms of the compound.

As used herein, the term “IC₅₀” refers to but not limited to theconcentration of a nucleotide analog at which its incorporation on aprimer-template complex yields equal numbers of moles of substrate andproduct and/or could be defined, but not limited to, incorporationefficiency measured by determining the concentration at which thecompound incorporates on half the primer-template complexes.

As used herein, the term “oligonucleotide” refers to DNA fragments of 2to 200 covalently linked nucleotides.

As used herein, the term “template” refers to an oligonucleotide servingas the complimentary strand for DNA synthesis (incorporation).

As used herein, the term “primer” refers to an oligonucleotide that ishybridized to a complement sequence on the template strand used toinitiate DNA synthesis (incorporation).

When used herein in the scientific or technical sense, the term“incorporation” refers to a nucleotide or nucleotide analog forming acomplement base-pair with the template strand and a covalent bond to aprimer strand by a polymerase. The primer-template complex is extendedone or more bases from the initial primer strand.

As used herein, the term “cleavage” refers to the removal of theterminating group by photo-cleavage, chemical cleavage, enzymaticcleavage or the like.

As used herein, the term “incorporation cycle” refers to theincorporation of a nucleotide or nucleotide analog by a polymerase, thedetection and identification of said nucleotide or nucleotide analog,and if a nucleotide analog, cleavage of the terminating group from saidanalog.

As used herein, the term “misincorporation” refers to a nucleotide ornucleotide analog forming a non-complement base-pair with the templatestrand and a covalent bond to a primer by a polymerase. Theprimer-template complex is extended one or more bases from the initialprimer strand.

As used herein, the term “discrimination” refers the IC₅₀ concentrationdifferences for misincorporation versus incorporation of nucleotide ornucleotide analogs by a polymerase.

As used herein, the term “termination” refers to the incorporation of anucleotide or nucleotide analog forming a complement or non-complementbase-pair with the template strand and a covalent bond to a primer by apolymerase. The primer-template complex is extended only one base fromthe initial primer strand for any given incorporation cycle.

As used herein, the term “DT₅₀” refers to the amount of time required tocleavage 50% of the base analog incorporated in the primer-templatecomplex.

The term “analog” as used herein, is understood as being a substancewhich does not comprise the same basic carbon skeleton and carbonfunctionality in its structure as a “given compound”, but which canmimic the given compound by incorporating one or more appropriatesubstitutions such as for example substituting carbon for heteroatoms.

An “isomer” of a first compound is a separate compound in which eachmolecule contains the same constituent atoms as the first compound, butwhere the configuration of those atoms in three dimensions differs.

As used herein, the term “patient” or “subject” refers to a livingmammalian organism, such as a human, monkey, cow, sheep, goat, dog, cat,mouse, rat, guinea pig, or transgenic species thereof. In certainembodiments, the patient or subject is a primate. Non-limiting examplesof human subjects are adults, juveniles, infants and fetuses.

When used in reference to a compound, composition, method or device,“pharmaceutically acceptable” means generally safe, non-toxic andneither biologically nor otherwise undesirable and includes that whichis acceptable for veterinary use as well as human pharmaceutical use.

As used herein, “predominantly one enantiomer” means that a compoundcontains at least about 85% of one enantiomer, or more preferably atleast about 90% of one enantiomer, or even more preferably at leastabout 95% of one enantiomer, or most preferably at least about 99% ofone enantiomer. Similarly, the phrase “substantially free from otheroptical isomers” means that the composition contains at most about 15%of another enantiomer or diastereomer, more preferably at most about 10%of another enantiomer or diastereomer, even more preferably at mostabout 5% of another enantiomer or diastereomer, and most preferably atmost about 1% of another enantiomer or diastereomer.

“Prevention” or “preventing” includes: (1) inhibiting the onset of adisease in a subject or patient which may be at risk and/or predisposedto the disease but does not yet experience or display any or all of thepathology or symptomatology of the disease, and/or (2) slowing the onsetof the pathology or symptomatology of a disease in a subject or patientwhich may be at risk and/or predisposed to the disease but does not yetexperience or display any or all of the pathology or symptomatology ofthe disease.

The term “saturated” when referring to an atom means that the atom isconnected to other atoms only by means of single bonds.

A “stereoisomer” or “optical isomer” is an isomer of a given compound inwhich the same atoms are bonded to the same other atoms, but where theconfiguration of those atoms in three dimensions differs. “Enantiomers”are stereoisomers of a given compound that are mirror images of eachother, like left and right hands. “Diastereomers” are stereoisomers of agiven compound that are not enantiomers.

The invention contemplates that for any stereocenter or axis ofchirality for which stereochemistry has not been defined, thatstereocenter or axis of chirality can be present in its R form, S form,or as a mixture of the R and S forms, including racemic and non-racemicmixtures.

“Therapeutically effective amount” or “pharmaceutically effectiveamount” means that amount which, when administered to a subject orpatient for treating a disease, is sufficient to effect such treatmentfor the disease.

“Treatment” or “treating” includes (1) inhibiting a disease in a subjector patient experiencing or displaying the pathology or symptomatology ofthe disease (e.g., arresting further development of the pathology and/orsymptomatology), (2) ameliorating a disease in a subject or patient thatis experiencing or displaying the pathology or symptomatology of thedisease (e.g., reversing the pathology and/or symptomatology), and/or(3) effecting any measurable decrease in a disease in a subject orpatient that is experiencing or displaying the pathology orsymptomatology of the disease.

As used herein, the term “water soluble” means that the compounddissolves in water at least to the extent of 0.010 mole/liter or isclassified as soluble according to literature precedence.

The above definitions supersede any conflicting definition in any of thereference that is incorporated by reference herein. The fact thatcertain terms are defined, however, should not be considered asindicative that any term that is undefined is indefinite. Rather, allterms used are believed to describe the invention in terms such that oneof ordinary skill can appreciate the scope and practice the presentinvention.

II. Synthetic Methods

Compounds of the present disclosure may be made using the methodsoutlined in the Examples section. These methods can be further modifiedand optimized using the principles and techniques of organic chemistryas applied by a person skilled in the art. Such principles andtechniques are taught, for example, in March's Advanced OrganicChemistry: Reactions, Mechanisms, and Structure (2007), which isincorporated herein by reference.

III. Biological Function and Cleavage Rates

As shown in some aspects of the present disclosure, substitution and itsstereochemistry of the α-carbon of the 2-nitrobenzyl group affectsbiological function and cleavage rates of reaction of 3′-unblocked,base-modified dNTPs.

In one embodiment, sample components enable the determination of SNPs.The method may be for the high-throughput identification of informativeSNPs. The SNPs may be obtained directly from genomic DNA material, fromPCR amplified material, or from cloned DNA material and may be assayedusing a single nucleotide primer extension method. The single nucleotideprimer extension method may comprise using single unlabeled dNTPs,single labeled dNTPs, single 3′-modified dNTPs, single base-modified3′-dNTPs, single alpha-thio-dNTPs or single labeled2′,3′-dideoxynucleotides. The mini-sequencing method may comprise usingsingle unlabeled dNTPs, single labeled dNTPs, single 3′-modified dNTPs,single base-modified 2′-dNTPs, single alpha-thio-dNTPs or single labeled2′,3′-dideoxynucleotides. The SNPs may be obtained directly from genomicDNA material, from PCR amplified material, or from cloned DNA materials.

The present disclosure further provides nucleotide and nucleosidecompounds as well as salts, esters and phosphates thereof, that can beused in rapid DNA sequencing technology. The compounds are optionally inthe form of ribonucleoside triphosphates (NTPs) and deoxyribonucleosidetriphosphates (dNTP). The nucleotide and nucleoside compounds in somecases include a photocleavable group labeled with a reporter group suchas a fluorescent dye. The nucleotide and nucleoside compounds includephotoremovable protecting groups that are designed to terminate DNAsynthesis as well as cleave rapidly, so that these monomers can be usedfor rapid sequencing in a parallel format. The presence of such rapidlycleavable groups labeled with fluorescent dyes on the nucleotide andnucleoside compounds can enhance the speed and accuracy of sequencing oflarge oligomers of DNA in parallel, to allow, for example, rapid wholegenome sequencing, and the identification of polymorphisms and othervaluable genetic information.

In certain aspects, the present disclosure relates to compounds whereinthe base of the nucleoside is covalently attached with a 2-nitrobenzylgroup, and the alpha carbon position of the 2-nitrobenzyl group isoptionally substituted with one alkyl or aryl group as described herein.In certain examples, the base of the nucleoside is covalently attachedwith a 2-nitrobenzyl group, and the 2-nitrobenzyl group is optionallysubstituted with one or more of an electron donating and electronwithdrawing group as described herein. The 2-nitrobenzyl group can befunctionalized to enhance the termination properties as well as thelight catalyzed deprotection rate. The termination properties of the2-nitrobenzyl and alpha carbon substituted 2-nitrobenzyl group attachedto the nucleobase occur even when the 3′-OH group on the ribose sugar isunblocked. These 3′-OH unblocked terminators are well-tolerated by anumber of commercially available DNA polymerases, representing a keyadvantage over 3′-O-blocked terminators. The alpha carbon substituted2-nitrobenzyl group also can be derivatized to include a selectedfluorescent dye or other reporter group.

A. Nucleotide and Nucleoside Compounds and their Use in DNA Sequencing

Nucleotide and nucleoside compounds are provided which are useful in DNAsequencing technology. One aspect of the present invention is directedtowards the use of the promising sequencing approach, cyclic reversibletermination (CRT). CRT is a cyclic method of detecting thesynchronistic, single base additions of multiple templates. Thisapproach differentiates itself from the Sanger method (Metzker, 2005,which is incorporated herein by reference) in that it can be performedwithout the need for gel electrophoresis, a major bottleneck inadvancing this field. Like Sanger sequencing, however, longerread-lengths translates into fewer sequencing assays needed to cover theentire genome. The CRT cycle typically comprises three steps,incorporation, imaging, and deprotection. For this procedure, cycleefficiency, cycle time, and sensitivity are important factors. The cycleefficiency is the product of deprotection and incorporation efficienciesand determines the CRT read-length. The CRT cycle time is the sum ofincorporation, imaging, and deprotection times. For rapid CRT for wholegenome sequencing, the nucleotide and nucleoside compounds as disclosedherein may be used, which can exhibit fast and efficient deprotectionproperties. These compounds can be labeled with reporter groups such asfluorescent dyes, attached directly to the 2-nitrobenzyl, providing,e.g., fluorescent, reversible terminators with similar deprotectionproperties. It has remained difficult to accomplish the goal of long CRTreads because reversible terminators typically act as poor substrateswith commercially available DNA polymerases. Modified nucleotide analogsof the present invention may be used to improve this technology byproviding substrates that incorporate as well or better than a naturalnucleotide with commercially available DNA polymerases.

When applied to genomic DNA, the compounds can be used in CRT to readdirectly from genomic DNA. Fragmented genomic DNA can be hybridized to ahigh-density oligonucleotide chip containing priming sites that spanselected chromosomes. Each priming sequence is separated by theestimated read-length of the CRT method. Between base additions, afluorescent imager can simultaneously image the entire high-densitychip, marking significant improvements in speed and sensitivity. Inspecific embodiments, a fluorophore, which is attached to the2-nitrobenzyl group or its derivatives described herein, is removed byUV irradiation releasing the 2-nitrobenzyl group for the next round ofbase addition. After approximately 500 CRT cycles, the complete andcontiguous genome sequence information can then be compared to thereference human genome to determine the extent and type of sequencevariation in an individual's sample. Reversible terminators that exhibithigher incorporation and deprotection efficiencies will typicallyachieve higher cycle efficiencies, and thus longer read-lengths.

CRT Efficiency is defined by the formula: (RL)^(Ceff)=0.5, where RL isthe read-length in bases and Ceff is the overall cycle efficiency. Inother words, a read-length of 7 bases could be achieved with an overallcycle efficiency of 90%, 70 bases could be achieved with a cycleefficiency of 99% and 700 bases with a cycle efficiency of 99.9%. Theefficiency of incorporation of compounds according to the invention mayrange from about 70% to about 100% of the incorporation of the analogousnative nucleoside. Preferably, the efficiency of incorporation willrange from about 85% to about 100%. Photocleavage efficiencies willpreferably range from about 85% to about 100%. Further, termination ofnucleic acid extension will range from about 90% to about 100% uponincorporation of compounds according to the invention. Nucleotide andnucleoside compounds in one embodiment have a cycle efficiency of atleast 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%,99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%.

Another aspect of the present invention is directed towards the use ofpyrosequencing, which is a non-electrophoretic, bioiluminescence methodthat measures the release of inorganic pyrophosphate (PPi) byproportionally converting it into visible light by a series of enzymaticreactions (Ronaghi et al., 1998, which is incorporated herein byreference). Unlike other sequencing approaches that use modifiednucleotides to terminate DNA synthesis, the pyrosequencing assaymanipulates DNA polymerase by the single addition of a dNTP in limitingamounts. DNA polymerase then extends the primer upon incorporation ofthe complementary dNTP and pauses. DNA synthesis is reinitiatedfollowing the addition of the next complementary dNTP in the dispensingcycle. The order and intensity of the light peaks are recorded asflowgrams, revealing the underlying DNA sequence. For homopolymerrepeats up to six nucleotides, the number of dNTPs added is directlyproportional to the light signal. Homopolymer repeats greater than sixnucleotide can result in insertional errors, which are the most commonerror type for pyrosequencing. Modified nucleotide analogs of thepresent invention may improve this technology by accurate sequencingthrough homopolymer repeats, particularly those greater than sixnucleotides in length.

Another aspect of the present invention is directed towards the use ofSanger sequencing, particularly in heterozygote detection. Despite muchadvancement, improvements in the dideoxy-BigDye terminator sequencingchemistry for accurate heterozygote detection are needed. It isgenerally believed that a uniform peak height distribution in theprimary data makes base-calling and heterozygote detection more reliableand accurate. The termination pattern in Sanger sequencing is primarilydue to sequence-dependent bias incorporation by DNA polymerase, whichcan selectively incorporate natural nucleotides over modifiednucleotides (Metzker et al., 1998, which is incorporated herein byreference). These bias incorporation effects are more pronounced withthe dye-terminator chemistry than with the dye-primer chemistry. Thiscan be attributed to effects of the large fluorescent dye structuresattached to the terminating nucleotide, lowering enzyme activity atleast 10-fold to that of the natural substrate. Thus, the reduction ofbias incorporation effects by DNA polymerase towards dye-labeledterminators could lead to improved heterozygote detection. Modifiednucleotide analogs of the present invention may improve this technologyby incorporating as well or better than a natural nucleotide, thuseliminating incorporation bias in Sanger sequencing.

Another aspect of the present invention is directed towards the use ofclonally amplified templates and single DNA molecule templates. Thefront-end of NGS technologies can be partitioned into two camps:clonally amplified templates from single DNA molecules and single DNAmolecule templates. It is well recognized in the art that DNA can beimmobilized to a solid surface by either attaching a primer to saidsurface and hybridizing a target nucleic acid to said primer (Southernand Cummins, 1998, U.S. Pat. No. 5,770,367; Harris et al., 2008, whichare incorporated herein by reference) or by attaching a target nucleicacid to said surface by clonally amplification and hybridizing a primerto said target nucleic acid (Dressman et al., 2003; Margulies et al.,2005, which are incorporated herein by reference). Either immobilizationconfiguration can be used in the present invention for then binding aDNA polymerase to initiate either the CRT method or the pyrosequencingmethod.

For CRT terminators to function properly, the protecting group must beefficiently cleaved under mild conditions. The removal of a protectinggroup generally involves either treatment with strong acid or base,catalytic or chemical reduction, or a combination of these methods.These conditions may be reactive to the DNA polymerase, nucleotides,oligonucleotide-primed template, or the solid support creatingundesirable outcomes. The use of photochemical protecting groups is anattractive alternative to rigorous chemical treatment and can beemployed in a non-invasive manner.

A number of photoremovable protecting groups including, but not limitedto 2-nitrobenzyl, benzyloxycarbonyl, 3-nitrophenyl, phenacyl,3,5-dimethoxybenzoinyl, 2,4-dinitrobenzenesulphenyl, and theirrespective derivatives have been used for the synthesis of peptides,polysaccharides, and nucleotides (Pillai, 1980, which is incorporatedherein by reference). Of these, the light sensitive 2-nitrobenzylprotecting group has been successfully applied to the 2′-OH ofribonucleosides for diribonucleoside synthesis (Ohtsuka et al., 1974,which is incorporated herein by reference), the 2′-OH ofribophosphoramidites in automated ribozyme synthesis (Chaulk andMacMillan, 1998, which is incorporated herein by reference), the 3′-OHof phosphoramidites for oligonucleotide synthesis in the Affymetrixchemistry (Pease et al., 1994, which is incorporated herein byreference), and to the 3′-OH group for DNA sequencing applications(Metzker et al., 1994, which is incorporated herein by reference). Underdeprotection conditions (ultraviolet light>300 nm), the 2-nitrobenzylgroup can be efficiently cleaved without affecting either the pyrimidineor purine bases (Pease et al., 1994 and Bartholomew and Broom, 1975,which are incorporated by reference).

In one aspect, the present invention is directed towards the use ofchemically cleavable reversible terminators. For example, the benzylprotecting group has been widely used in organic synthesis as a resultof its stability and ease of mild and selective deprotection bycatalytic hydrogenolysis (Green and Wuts, 1999, which is incorporatedherein by reference). Hydrogenolysis, which can be conducted underneutral conditions, is advantageous when working with nucleosidescontaining phosphoanhydride bonds, since nucleoside diphosphates, andespecially nucleoside triphosphates, degrade under acidic conditions (Wuet al., 2004; Johnson et al., 2004, which are incorporated herein byreference). Removal of a benzyl protecting group from solid-supportedcompounds by hydrogenolysis using Palladium nano-particles (Kanie etal., 2000, which is incorporated herein by reference) and hydrogenationconducted on a microfluidic device with immobilized Palladium catalyst(Kobayashi et al. 2004, which is incorporated herein by reference) havealso been reported in addition to hydrogenolysis using conventionalPalladium catalyst.

B. Polymerase Assays

Natural and modified nucleotides were tested for incorporationefficiency using the “polymerase end point assay” (Wu et al., 2007,which is incorporated herein by reference). This assay examinesincorporation efficiency on matched and mismatched template bases.Incorporation efficiency is measured by determining the concentration atwhich the compound incorporates on half the primer-template complexes(IC₅₀). Titrations of increasing compound concentration were performedto generate curves from which the IC₅₀ can be determined.

The sequence of the template DNA is selected depending on which compoundwill be tested. For example, the first interrogation base after theprimer in the template sequence is the complement base of the compoundwhen measuring incorporation efficiency, and one of three mismatchedbases when measuring mismatch discrimination properties.

To the annealed reaction, a DNA polymerase (e.g., THERMINATOR™ DNApolymerase, 0.25 units per reaction, New England Biolabs), 1× ThermopolBuffer, and a known concentration of either natural or modifiednucleotide are added to each 10 μL reaction and incubated at 75° C. for10 minutes, cooled on ice, and quenched with 10 μL of stop solution (98%formamide: 10 mM Na₂EDTA, pH=8.0, 25 mg/ml Blue Dextran). Stoppedreactions are heated to 75° C. for 30 seconds to denature the DNA, andthen placed on ice. The extension products are analyzed on a 10% LongRanger (Lonza) polyacrylamide gel using an ABI model 377 DNA sequencer.Additional details are provided in Example 1, below.

FIG. 1 compares the incorporation with THERMINATOR™ DNA polymerase ofnucleotides disclosed herein with natural nucleotides. For example,compound 3p065 (5-(α-isopropyl-2-nitrobenzyloxymethyl-2′-dCTP) reaches50% incorporation at a lower concentration than its natural analog(IC₅₀=1.1±0.1 nM versus 3.0±0.6 nM). Compounds labeled with dye alsoincorporate efficiency with THERMINATOR™ DNA polymerase, for examplecompound 6p038/6p017 5-(α-isopropyl-2-nitrobenzyl-oxy)methyl-2′-dCTP-Cy5has an IC₅₀=5.1±1.4 nM. Table A shows the IC₅₀ concentrations for manyof the modified nucleotide analogs described herein.

TABLE A Effects of Substitution at the α-Carbon on MismatchDiscrimination. WW# Chemical Name Diastereomer IC₅₀ for incorporation1p129 N⁶-(2-nitrobenzyl)-2′-dATP 2.5 ± 0.3 nM 2p0435-(α-methyl-nitrobenzyl-oxymethyl)-2′-dUTP mixture 1.7 ± 0.2 nM 2p108O⁶-(2-nitrobenzyl)-2′-dGTP 4.0 ± 0.7 nM 2p143O⁶-(α-methyl-2-nitrobenzyl)-2′-dGTP mixture 10 ± 0.0 nM 2p1485-(α-isopropyl-nitrobenzyl-oxymethyl)-2′-dUTP mixture 2.3 ± 0.1 nM 3p006N⁶-(α-methyl-2-nitrobenzyl)-2′-dATP mixture 8.5 ± 1.8 nM 3p0635-(α-isopropyl-2-nitrobenzyl-oxymethyl)-2′-dUTP single 2.0 nM 3p0655-(α-isopropyl-2-nitrobenzyl-oxymethyl)-2′-dCTP single 1.1 ± 0.1 nM3p075 5-(α-tert-butyl-2-nitrobenzyl-oxymethyl)-2′-dUTP single 3.0 nM3p085 5-(α-tert-butyl-2-nitrobenzyl-oxymethyl)-2′-dCTP single 2.0 nM5p085 C⁷-(2-nitrobenzyl-oxymethyl)-2′-dATP 3.6 ± 0.3 nM 5p098-ds1C⁷-(α-isopropyl-2-nitrobenzyl-oxymethyl)-2′-dATP (ds1) single 6.7 ± 0.8nM 5p098-ds2 C⁷-(α-isopropyl-2-nitrobenzyl-oxymethyl)-2′-dATP (ds2)single 5.7 ± 1.1 nM 5p107 C⁷-(2-nitrobenzyl-oxymethyl)-2′-dGTP 1.5 ± 0.3nM 5p111 5-(α-isopropyl-benzyl-oxymethyl)-2′-dUTP mixture 0.8 nM 5p127C⁷-(α-isopropyl-2-nitrobenzyl-oxymethyl)-2′-dATP-6-FAM single 6.4 ± 1.0nM 5p130-LP2 C⁷-(α-isopropyl-2-nitrobenzyl-oxymethyl)-2′-dATP-6-CR110single 15 ± 3 nM 5p143-ds1C⁷-(α-isopropyl-2-nitrobenzyl-oxymethyl)-2′-dGTP (ds1) single 1.2 ± 0.2nM 5p143-ds2 C⁷-(α-isopropyl-2-nitrobenzyl-oxymethyl)-2′-dGTP (ds2)single 1.3 ± 0.3 nM 5p145 5-benzyl-oxymethyl-2′-dUTP 1.4 nM 5p1475-(2-methyl-benzyl-oxymethyl)-2′-dUTP 1.4 nM 5p1495-(2-isopropyl-benzyl-oxymethyl)-2′-dUTP 1.4 nM 6p0055-(α-isopropyl-2-nitrobenzyl-oxymethyl)-2′-dUTP-5-R6G single 15 ± 1 nM6p008 5-(α-isopropyl-2-nitrobenzyl-oxymethyl)-2′-dUTP-6-JOE single 9.0 ±1.4 nM 6p010 5-(2-phenyl-benzyl-oxymethyl)-2′-dUTP 1.4 nM 6p0155-(2,6-dimethyl-benzyl-oxymethyl)-2′-dUTP 1.4 nM 6p0175-(α-isopropyl-2-nitrobenzyl-oxymethyl)-2′-dCTP-Cy5 single 4.8 ± 1.9 nM6p024 5-(2-tert-butyl-benzyl-oxymethyl)-2′-dUTP 0.7 nM 6p028/5p127C⁷-(α-isopropyl-2-nitrobenzyl-oxymethyl)-2′-dATP-6-FAM single 6.4 ± 1.0nM 6p034 C⁷-(α-isopropyl-2-nitrobenzyl-oxymethyl)-2′-dGTP-6-ROX single7.4 ± 0.1 nM 6p038/6p0175-(α-isopropyl-2-nitrobenzyl-oxymethyl)-2′-dCTP-Cy5 single 5.1 ± 1.4 nM6p044 5-(α-isopropyl-2-nitrobenzyl-oxymethyl)-2′-dUTP-6-ROX single 18 ±1 nM 6p057-ds1 C⁷-(α-isopropyl-2,6-di-nitrobenzyl-oxymethyl)-2′-dATP(ds1) single 5.2 ± 0.2 nM 6p057-ds2C⁷-(α-isopropyl-2,6-di-nitrobenzyl-oxymethyl)-2′-dATP (ds2) single 5.8 ±1.1 nM 6p063 N⁶-(α-isopropyl-2-nitrobenzyl)-2′-dATP single 12 ± 1 nM6p075/6p008 5-(α-isopropyl-2-nitrobenzyl-oxymethyl)-2′-dUTP-6-JOE single9.0 ± 1.4 nM 6p087-ds1C⁷-(α-isopropyl-4-methoxy-2-nitrobenzyl-oxymethyl)-2′-dATP (ds1) single15 ± 1 nM 6p087-ds2C⁷-(α-isopropyl-4-methoxy-2-nitrobenzyl-oxymethyl)-2′-dATP (ds2) single18 ± 3 nM 6p094 5-(α-isopropyl-2-nitrobenzyl-oxymethyl)-2′-dUTP-6-FAMsingle 8.1 ± 0.2 nM

C. Mismatch Discrimination

It has been reported that substitution at the α-carbon of the2-nitrobenzyl group can increase the rate of the cleavage reaction(Reichmanis et al., 1985; Cameron and Frechet, 1991; Hasan et al., 1997,which are incorporated herein by reference). Without being bound bytheory, the results presented herein suggest that substitution at theα-carbon of the 2-nitrobenzyl group can also affect the termination ofDNA synthesis for 3′-unblocked nucleotide triphosphates and improvediscrimination against mismatch incorporation. Furthermore, and based onthe results discussed in greater detail below, it was found that thestereochemistry of the substitution of α-carbon of the 2-nitrobenzylgroup can have a significant impact on the extent of mismatchdiscrimination and the rate of the cleavage reaction.

Table 1 shows a comparison between the two diastereomers “ds1” and “ds2”of the α-substituted carbon and their precursors. The mismatch/matchratios represent the ability of the polymerase to distinguish betweencorrectly incorporating against a matched template base and a mismatchone. Discrimination is considered sufficient when the mismatch/matchratio is greater than or equal to 100 (two orders of magnitude). Table 1shows the discrimination of natural dGTP and C⁷-hydroxymethyl-dGTPanalogs 5p107, 5p143-ds1 and 5p143-ds2. Both natural dGTP andC⁷-(2-nitrobenzyloxy)methyl-2′-dGTP (5p107) show mismatch/match ratiosranging from 10 to 360, and 10 to 250, respectively, two of which arebelow the 100-fold threshold. Substitution of the α-methylene carbonwith an isopropyl group, however, results in a significantly higherdiscrimination ratio for all mismatch template bases. 5p143-ds1 exhibitsa high mis-match discrimination ratio compared with 5p143-ds2. Thesedata provide evidence that the stereochemistry of the α-isopropyl groupcan affect the degree of discrimination against mismatch incorporation.A similar trend is observed for the C⁷-hydroxymethyl-dATP analogs. Forthe compounds shown in Table 1, substitution at the α-carbon of the2-nitrobenzyl group increases mismatch discrimination in astereo-specific manner.

TABLE 1 Effects of Substitution at the α-Carbon on MismatchDiscrimination. Complement Mismatch/match ratio (10²) Base dGTP 5p1075p143-ds1 5p143-ds2 “C” N/A N/A N/A N/A “T” 0.1 0.1 5 2.5 “A” 3.62.5 >830 >770 “G” 0.2 0.4 >21 >7.7 dGTP = natural dGTP 5p107 =C⁷-(2-nitrobenzyloxy)methyl-2′-dGTP 5p143 =C⁷-(α-isopropyl-2-nitrobenzyloxy)methyl-2′-dGTP

D. Termination

Without being bound by theory, at least two factors were found totypically influence termination of DNA synthesis after a singleincorporation: a) substitution at the α-carbon of the 2-nitrobenzylgroup, and b) substitution at the 2-position of the benzyl ring. Table 2shows the influence of various substitutions using a “weighted sum”analysis, which is determined by quantifying primer extension productsusing automated gel electrophoresis. A weighted sum of 1.0 representscomplete termination after a single incorporation, while a value greaterthan 1.0 indicates incorporation beyond the +1 position (e.g. nucleotideread through). To standardize the termination assay, a concentration of25× the IC₅₀ value of a given compound is used. The assay is performedas described above for the polymerase end-point assay, except that thetemplate used is a homopolymer repeat, thereby allowing for multipleincorporations of a given nucleotide compound. In this example, modifieddUTP analogs are compared to natural dTTP, which at 25× its IC₅₀ valueextends the entire length of the homopolymer repeat template andmisincorporates the 11^(th) base (weighted sum=11). Compound 3p085(2-nitrobenzyloxymethyl-2′-dUTP) shows a degree of termination with aweighted sum of 3.7±0.1. Substitution of the α-carbon with a methylgroup (2p043) further improves termination, reducing the weighted sumvalue to 1.7±0.1. Complete termination is achieved with an α-isopropylsubstitution (2p148), showing a weighted sum value of 1.0. The IC₅₀value for the complement base, however, does not increase, indicatingthat the larger isopropyl substitution has a beneficial effect ontermination, but does not compromise incorporation efficiency.

TABLE 2 Both substitutions at the α-carbon of the 2-nitrobenzyl ring,and substitution at the 2- position of the benzyl ring influencetermination properties of the compound Adj IC₅₀ Average Weighted Sum ±SD Compound name Substitution Conc (nM) 1x IC₅₀ 5x IC₅₀ 25x IC₅₀ dTTP(natural dTTP) 2.1 0.68 3.0 12.2 2p043 α-methyl-5-(2-nitrobenzyl) 1.70.53 ± 0.06 1.1 ± 0.0 1.7 ± 0.1 2p148 α-isopropyl-5-(2-nitrobenzyl) 2.10.59 ± 0.02 0.99 ± 0.01  1.0 ± 0.01 5p111 α-isopropyl-5-benzyl 0.8 0.44± 0.02 0.96 ± 0.01  1.1 ± 0.03 5p145 5-benzyl 1.4 0.43 ± 0.1 1.3 ± 0.32.9 ± 0.4 5p147 5-(2-methyl benzyl) 1.4 0.51 ± 0.1 1.5 ± 0.3 2.6 ± 0.45p149 5-(2-isopropyl benzyl) 1.4 0.53 ± 0.1 1.3 ± 0.1 2.2 ± 0.2 6p0105-(2-phenyl benzyl) 1.4 0.46 ± 0.1 1.1 ± 0.1 1.7 ± 0.3 6p0155-(2,6-dimethyl benzyl) 1.4 0.56 ± 0.1 1.5 ± 0.1 2.2 ± 0.1 6p0245-(2-tertbutyl benzyl) 0.7 0.44 ± 0.1 1.2 ± 0.1 1.9 ± 0.1

The substitution of the 2-position on the benzyl ring can also influencethe termination properties of these modified nucleotides. For example,removing the nitro group from this position (compound 5p111) increasesthe weighted sum value to 1.1 for an α-isopropyl substitution analog. Anumber of dUTP analogs were synthesized with various substituents on the2-position and characterized for termination in the absence of theisopropyl group at the α-carbon. Table 2 shows a general trend ofincreasing substituent size and shape and improved terminationproperties, compare 5p145 (WS=2.9) and 6p010 (WS=1.7) at 25×IC₅₀concentrations.

E. UV-Cleavage Rates

Cleavage of the terminating substituted 2-nitrobenzyl group when analogsare incorporated into the primer strand with 365 nm UV light allows forthe next cycle of incorporation to resume. Without being bound bytheory, at least two factors were found typically influence UV-cleavagerates of incorporated nucleotide analogs: a) stereo-chemistry of theα-carbon substitution of the 2-nitrobenzyl group, and b) substitution onthe benzyl ring. Incorporation on a matched template is performed asdescribed above using 1 μM concentration to extend the primer strand.Ten identical tubes are used for each incorporation experiment, afterwhich NaN₃ is added to a final concentration of 50 mM. Extended primerreactions are exposed to 365 nm light for various time points using theUV deprotector device described by Wu et al. (2007, which isincorporated herein by reference) and analyzed using an AB model 377 DNAsequencer. The quantitative data are plotted linearly as productformation versus exposure time, and the time point at which half thenucleotide analog is cleaved is calculated, as a DT₅₀ value. As shown inTable 3, the stereochemistry affects the rate of UV-cleavage forC⁷-hydroxymethyl-dATP analogs 5p098-ds1 and -ds2, 6p057-ds1 and -ds2,and 6p087-ds1 and -ds2. For example, based on these examples, the ds2analogs show faster cleavage rates (e.g., lower DT₅₀ values) comparedwith ds1 analogs. Furthermore, substitution of a methoxy group at the 4position on the benzyl ring further increases the rate of UV-cleavage,albeit for ds2 analogs only (e.g., the DT₅₀ value for 5p098-ds2 is 3.3sec versus that of 1.9 sec for 6p087-ds2). Based on the examplessummarized in Table 3, substitution at the α-carbon of the 2-nitrobenzylring increases UV-cleavage in a stereospecific manner.

TABLE 3 Table 3, UV-Cleavage Rates of 2-Nitrobenzyl Ring Derivatives.Compound Substitution DT₅₀ (seconds) 5p098-ds1 α-isopropyl-2- 7.5 ± 0.85p098-ds2 nitrobenzyl 3.3 ± 0.2 6p057-ds1 α-isopropyl-2,6- 7.3 ± 1.26p057-ds2 nitrobenzyl 4.8 ± 0.5 6p087-ds1 α-isopropyl-2- 8.8 ± 0.66p087-ds2 nitrobenzyl-4- 1.9 ± 0.1 methoxy

F. Chemical cleavage

In one aspect, the present invention is directed towards the use ofchemically cleavable reversible terminators. For example, the benzylprotecting group has been widely used in organic synthesis as a resultof its stability and ease of mild and selective deprotection bycatalytic hydrogenolysis (Green and Wuts, 1999, which is incorporatedherein by reference). Hydrogenolysis, which can be conducted underneutral conditions, is advantageous when working with nucleosidescontaining phosphoanhydride bonds, since nucleoside diphosphates, andespecially nucleoside triphosphates, degrade under acidic conditions (Wuet al., 2004; Johnson et al., 2004, which are incorporated herein byreference). Removal of a benzyl protecting group from solid-supportedcompounds by hydrogenolysis using palladium nano-particles (Kanie etal., 2000, which is incorporated herein by reference) and hydrogenationconducted on microfluidic device with immobilized palladium catalyst(Kobayashi et al. 2004, which is incorporated herein by reference) havealso been reported beside hydrogenolysis using conventional palladiumcatalyst. Examples of chemically reversible cleavage results, includingcleavage of 5-benzyloxymethyl-dU analogs using catalytic hydrogenolysis,are provided in Example 10 below.

IV. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Methods and Materials

Polymerase Assays. Natural and modified nucleotides were tested forincorporation efficiency using the “polymerase end point assay” (Wu etal., 2007, which is incorporated herein by reference). This assayexamines incorporation efficiency on matched and mismatched templatebases. Incorporation efficiency is measured by determining theconcentration at which the compound incorporates on half theprimer-template complexes (IC₅₀). Titrations of increasing compoundconcentration were performed to generate curves from which the IC₅₀ canbe determined.

The assay is performed by first annealing 5 nM of BODIPY-FL labeledprimer with 40 nM template DNA in 1× Thermopol Buffer (20 mM Tris-HCl,pH 8.8; 10 mM (NH₄)₂SO₄; 10 mM KCl; 2 mM MgSO₄; 0.1% Triton X-100, NewEngland BioLabs).

The temperature cycle to complete primer annealing is 80° C. for 30seconds, 57° C. for 30 seconds, then cooling to 4° C. The sequence ofthe template DNA is selected depending on which compound will be tested.For example, the first interrogation base after the primer in thetemplate sequence is the complement base of the compound when measuringincorporation efficiency, and one of three mismatched bases whenmeasuring mismatch discrimination properties.

To the annealed reaction, a DNA polymerase (e.g., THERMINATOR™ DNApolymerase, 0.25 units per reaction, New England Biolabs), 1× ThermopolBuffer, and a known concentration of either natural or modifiednucleotide are added to each 10 μL reaction and incubated at 75° C. for10 minutes, cooled on ice, and quenched with 10 μL of stop solution (98%formamide: 10 mM Na₂EDTA, pH=8.0, 25 mg/ml Blue Dextran). Stoppedreactions are heated to 75° C. for 30 seconds to denature the DNA, andthen placed on ice. The extension products are analyzed on a 10% LongRanger (Lonza) polyacrylamide gel using an ABI model 377 DNA sequencer.The quantitative data are displayed as a linear-log plot of productformation versus compound concentration, and the IC₅₀ is calculatedusing KaleidaGraph software (Synergy Software).

Example 2 Synthesis of α-Substituted 2-Nitrobenzyl Alcohols Synthesis of(RS)-1-(2-nitrophenyl)ethanol

(RS)-1-(2-Nitrophenyl)ethanol: Sodium borohydride (0.69 g, 18.16 mmol)was added to a solution of a 2-nitroacetophenone (1.0 g, 6.06 mmol) inmethanol (9 mL) and 1,4-dioxane (6 mL) in small portions (Dong et al.,2005, which is incorporated herein by reference). The mixture wasstirred at room temperature for 30 minutes, then concentrated in vacuo.The residue was diluted with acetyl acetate (50 mL), washed with water(10 mL) and brine (10 mL). The organic phase was dried over Na₂SO₄ andconcentrated in vacuo to yield racemic (RS)-1-(2-nitrophenyl)ethanol(1.02 g, 100%). ¹H NMR (400 MHz, CDCl₃): δ 7.90 (m, 1 H, Ph-H), 7.84 (m,1 H, Ph-H), 7.66 (m, 1 H, Ph-H), 7.44 (m, 1 H, Ph-H), 5.42 (m, 1 H,Ph-CH), 2.33 (d, 1 H, J=3.5 Hz, OH) 1.58 (d, 3 H, J=5.1 Hz, CH₃).

Synthesis of (RS)-1-(4-iodo-2-nitrophenyl)ethanol

4-Iodo-2-nitrotoluene: To a suspension of 4-methyl-3-nitroaniline (4.30g, 28.26 mmol) in water (40 mL) cooled in ice-water bath, 98% sulfuricacid (1.89 mL) was added cautiously (Herm et al., 2002, which isincorporated herein by reference). Sodium chloride was added into theice-water bath to lower the temperature to minus 2° C., and a solutionof NaNO₂ (2.15 g, 31.10 mmol) in water (10 mL) was added at a rate thatthe reaction temperature did not exceed 0° C. Upon completion of theaddition, the mixture was stirred at minus 2° C. for 45 minutes. Thissolution of the diazo compound was then carefully added (in smallportions) to a boiling solution of NaI (12.89 g, 86 mmol) (CAUTION:vigorous gas evolution). Upon completion of the addition, the reactionmixture was cooled down to room temperature and extracted with methylenechloride (50 mL) four times. The combined organic phase was washed withsaturated NaHCO₃ (40 mL) and water (40 mL), dried over Na₂SO₄,concentrated in vacuo. The residue was purified by silica gelchromatography to yield 4-iodo-2-nitrotoluene (6.07 g, 82%). ¹H NMR (400MHz, CDCl₃): δ 8.28 (d, 1 H, J=1.8 Hz, Ph-H), 7.81 (dd, 1 H, J=2.2 and8.1 Hz, Ph-H), 7.09 (d, 1 H, J=8.1 Hz, Ph-H), 2.55 (s, 3 H, CH₃).

1-Bromomethyl-4-iodo-2-nitrobenzene: NBS (5.45 g, 30.62 mmol) andbenzoyl peroxide (75% aq, 200 mg, 0.85 mmol) were added to a solution of4-iodo-2-nitrotoluene (4.63 g, 17.60 mmol) in CCl₄ (60 mL). The mixturewas heated to reflux overnight, then cooled to room temperature,concentrated in vacuo, and purified by column chromatography to yield1-bromomethyl-4-iodo-2-nitrobenzene (2.71 g, 45%). ¹H NMR (400 MHz,CDCl₃): δ 8.35 (d, 1 H, J=1.8 Hz, Ph-H), 7.93 (dd, 1 H, J=1.8 and 8.1Hz, Ph-H), 7.30 (d, 1 H, J=8.1 Hz, Ph-H), 4.76 (s, 2 H, PhCH₂).

4-Iodo-2-nitrobenzaldehyde: NaHCO₃ (3.32 g, 39.48 mmol) was added to asolution of 1-bromomethyl-4-iodo-2-nitrobenzene (2.25 g, 6.58 mmol) inanhydrous DMSO (150 mL). The mixture was stirred at 86° C. for 19 hoursunder a nitrogen atmosphere, then cooled down to room temperature,diluted with water (300 mL), and extracted with methylene chloride fourtimes (250 mL). The combined organic phase was dried over Na₂SO₄,concentrated in vacuo, and purified by silica gel chromatography toyield 4-iodo-2-nitrobenzaldehyde (1.11 g, 61%). ¹H NMR (400 MHz, CDCl₃):δ 10.38 (d, 1 H, J=1.6 Hz, CHO), 8.45 (d, 1 H, J=1.5 Hz, Ph-H), 8.15(dd, 1 H, J=1.5 and 8.0 Hz, Ph-H), 7.67 (d, 1 H, J=8.1 Hz, Ph-H); ¹³CNMR (100 MHz, CDCl₃): δ 187.28 (CH), 143.18 (CH), 133.29 (CH), 130.67(CH), 130.28 (C), 109.60 (C), 99.78 (C).

(RS)-1-(4-Iodo-2-nitrophenyl)ethanol: Titanium(IV) isopropoxide (1.2 mL,4.04 mmol) was dissolved in anhydrous dichloromethane (8 mL) under anitrogen atmosphere. The solution was cooled to 0° C. and dimethylzinc(2 M in toluene, 8.67 mL, 17.34 mmol) was added dropwise. The mixturewas stirred at 0° C. for 45 minutes followed by addition of4-iodo-2-nitrobenzaldehyde (800 mg, 2.89 mmol). The mixture was stirredat 0° C. for 36 hours, then quenched by 1 M HCl (CAUTION: vigorous gasevolution!) and extracted with ethyl either (50 mL) three times. Thecombined organic phase was washed with water (50 mL), saturated NaHCO₃solution (50 mL) and brine (50 mL), dried over Na₂SO₄, concentrated invacuo, and purified by column chromatography to yield racemic(RS)-1-(4-Iodo-2-nitrophenyl)ethanol (408 mg, 48%). ¹H NMR (400 MHz,CDCl₃): δ 8.21 (dd, 1 H, J=1.8 and 5.3 Hz, Ph-H), 7.96 (m, 1 H, Ph-H),7.58 (d, 1 H, J=8.3 Hz, Ph-H), 5.38 (q, 1 H, J=6.1 Hz, Ph-CH), 2.34 (brs, 1 H, OH), 1.54 (d, 3 H, J=6.1 Hz, CH₃); ¹³C NMR (100 MHz, CDCl₃): δ147.98 (C), 142.49 (CH), 140.68 (C), 132.77 (CH), 131.31 (CH), 91.60(C), 65.40 (CH), 24.28 (CH₃).

Synthesis of (RS)-1-(2-nitrophenyl)-2-methyl-1-propanol and(S)-1-(2-nitrophenyl)-2-methyl-1-propanol

(RS)-1-(2-Nitrophenyl)-2-methyl-propanol: To a solution of1-iodo-2-nitrobenzene (1.12 g, 4.5 mmol) in anhydrous THF (15 mL) cooledto minus 40° C. under nitrogen atmosphere, a solution of phenylmagnesiumchloride (2 M in THF, 2.4 mL, 4.8 mmol) was added dropwise at a ratethat the temperature would not exceed minus 35° C. Upon completion ofthe addition the mixture was stirred for five minutes at minus 40° C.,followed by addition of isobutyraldehyde (0.545 mL, 6.0 mmol). Themixture was gradually warmed up to room temperature, quenched withsaturated ammonium chloride (6 mL), poured into water (80 mL), andextracted with ethyl acetate three times (100 mL). Combined organicphase was washed with brine (80 mL), dried over Na₂SO₄, and concentratedin vacuo. The residue was purified by silica gel column chromatographyto yield racemic (RS)-1-(2-nitrophenyl)-2-methyl-propanol (0.876 g, 99%)as a light yellow oil. ¹H NMR (400 MHz, CDCl₃): δ 7.84 (dd, 1 H, J=1.2and 8.2 Hz, Ph-H), 7.73 (dd, 1 H, J=1.4 and 7.9 Hz, Ph-H), 7.61 (m, 1 H,Ph-H), 7.39 (m, 1 H, Ph-H), 5.03 (dd, 1 H, J=4.5 and 5.8 Hz, Ph-CH),2.42 (d, 1 H, J=4.5 Hz, OH), 2.02 (m, 1 H, CH), 0.95 (d, 3 H, J=6.7 Hz,CH₃), 0.89 (d, 3 H, J=6.8 Hz, CH₃); ¹³C NMR (100 MHz, CDCl₃): δ 148.48(C), 138.91 (C), 132.95 (CH), 128.85 (CH), 127.95 (CH), 124.21 (CH),73.79 (CH), 34.30 (CH), 19.66 (CH₃), 17.01 (CH₃).

(S)-1-(2-Nitrophenyl)-2-methyl-propyl (1S)-camphanate: To a solution of(RS)-1-(2-nitrophenyl)-2-methyl-propanol (2.18 g, 11.2 mmol) inanhydrous pyridine (10 mL) (1S)-camphanic acid chloride (2.63 g, 12.2mmol) was added. The mixture was stirred for 18 hours at roomtemperature under nitrogen atmosphere, then concentrated in vacuo toafford crude (RS)-1-(2-nitrophenyl)-2-methyl-propyl (1S)-camphanate (1:1mixture of diastereomers). The camphanate was dissolved in boilingmethanol (100 mL) and the solution was allowed to cool to roomtemperature and left overnight. Crystals formed were collected byfiltration and were redissolved in boiling methanol (80 mL) and thesolution was allowed to cool to room temperature and left overnight.Crystals formed were collected by filtration to afford of pure(S)-1-(2-nitrophenyl)-2-methyl-propyl (1S)-camphanate (0.76 g, 36%). ¹HNMR (400 MHz, CDCl₃): δ 7.99 (dd, 1 H, J=1.8 and 7.8 Hz, Ph-H), 7.63 (m,2 H, Ph-H), 7.45 (m, 1 H, Ph-H), 6.33 (d, 1 H, J=6.0 Hz, Ph-CH), 2.32(m, 2 H), 1.91 (m, 2 H), 1.67 (m, 1 H), 1.12 (s, 3H, CH₃), 1.05 (s, 3 H,CH₃), 1.03 (d, 3 H, J=6.8 Hz, CH₃), 1.00 (s, 3 H, CH₃), 0.99 (d, 3 H,J=6.8 Hz, CH₃).

X-ray Crystallography data of (S)-1-(2-nitrophenyl)-2-methyl-propyl(1S)-camphanate: C₂₀H₂₅NO₆, M=375.41, colorless plate, 0.26×0.24×0.10mm³, orthorhombic, space group P2₁2₁2₁ (No. 19), a=11.9268(15),b=11.9812(14), c=13.5488(16) Å, V=1936.1(4) Å³, Z=4, D_(c)=1.288 g/cm³,F₀₀₀=800, MWPC area detector, CuKα radiation, λ=1.54178 Å, T=110(2)K,2θ_(max)=120.0°, 22896 reflections collected, 2665 unique(R_(int)=0.0462). Final GooF=1.009, R1=0.0219, wR2=0.0554, R indicesbased on 2629 reflections with I>2sigma(I) (refinement on F²), 245parameters, 0 restraints. Lp and absorption corrections applied μ=0.787mm⁻¹. Absolute structure parameter=0.09(5) (Flack, 1983, which isincorporated herein by reference). See FIG. 2.

(S)-1-(2-Nitrophenyl)-2-methyl-propanol:(S)-1-(2-Nitrophenyl)-2-methyl-propyl (1S)-camphanate (0.717 g, 1.90mmol) was dissolved in hot methanol (40 mL) and K₂CO₃ (0.380 g, 2.74mmol) was added. The mixture was heated to reflux for one hour, thencooled down, concentrated in vacuo, and diluted with diethyl ether (100mL). The organic phase was washed with water (20 mL), dried overanhydrous Na₂SO₄, and purified by silica gel column chromatography toyield (S)-1-(2-nitrophenyl)-2-methyl-propanol (0.360 g, 97%) as a lightyellow oil. ¹H NMR was identical to that of the racemic alcohol.

Synthesis of (RS)-1-(4-Iodo-2-nitrophenyl)-2-methyl-1-propanol,(R)-1-(4-Iodo-2-nitrophenyl)-2-methyl-1-propanol and(S)-1-(4-Iodo-2-nitrophenyl)-2-methyl-1-propanol

1,4-Diiodo-2-nitrobenzene 4-Iodo-2-nitroaniline (6.60 g, 0.025 mol) wassuspended in water (19 mL) and glacial acetic acid (17.5 mL) (Sapountziset al., 2005, which is incorporated herein by reference). The mixturewas cooled to 0° C. Sulfuric acid (17.5 mL, 0.328 mol) was addedcautiously. The mixture was cooled to minus 5° C., and a solution ofNaNO₂ (1.90 g, 0.028 mol) in water (7.5 mL) was added dropwise at a ratethat the temperature would not exceed 0° C. Upon completion of theaddition the mixture was stirred for 30 minutes and was added in smallportions to a boiling solution of sodium iodide (22.33 g, 0.149 mol) inwater (7.5 mL) (CAUTION: vigorous nitrogen evolution!). The resultingmixture was kept at 60° C. for one hour, then cooled down to roomtemperature, followed by addition of diethyl ether (500 mL). The ethersolution was separated, washed twice with water (150 mL) and oncesaturated NaHCO₃ (150 mL). The solution was dried over Na₂SO₄ andconcentrated in vacuo to a solid, which was recrystallized from ethanolto yield 1,4-diiodo-2-nitrobenzene (9.30 g, 99%). ¹H NMR (400 MHz,CDCl₃): δ 8.15 (d, 1H, J=2.0 Hz), 7.75 (d, 1 H, J=8.3 Hz), 7.56 (dd, 1H, J=8.3 and 2.0 Hz).

(RS)-1-(4-Iodo-2-nitrophenyl)-2-methyl-1-propanol: To a solution of1,4-diiodo-2-nitrobenzene (2.24 g, 6.0 mmol) in anhydrous THF (20 mL) atminus 40° C. under a nitrogen atmosphere, phenylmagnesium bromide (2 Min THF, 3.2 mL, 6.4 mmol) was added dropwise at a rate that thetemperature would not exceed minus 35° C. Upon completion of theaddition the mixture was stirred for 5 minutes, followed by addition ofisobutyraldehyde (0.726 mL, 8.0 mmol). The mixture was gradually warmedup to room temperature, quenched with saturated NH₄Cl (8 mL), thenpoured into water (100 mL). The mixture was extracted three times withethyl acetate (150 mL), and the combined organic phase was washed withbrine (100 mL), dried over Na₂SO₄, concentrated in vacuo and purified bysilica gel column chromatography to yield racemic(RS)-1-(4-Iodo-2-nitrophenyl)-2-methyl-1-propanol (1.54 g, 80%) as alight yellow oil. ¹H NMR (400 MHz, CDCl₃): δ 8.18 (d, 1 H, J=1.7 Hz,Ph-H), 7.93 (dd, 1 H, J=1.7 and 8.3 Hz, Ph-H), 7.50 (d, 1 H, J=8.3 Hz,Ph-H), 5.04 (m, 1 H, Ph-CH), 2.17 (d, 1 H, J=4.4 Hz, OH), 1.98 (m, 1 H,CH), 0.93 (d, 3 H, J=4.4 Hz, CH₃), 0.92 (d, 3 H, J=4.4 Hz, CH₃); ¹³C NMR(100 MHz, CDCl₃): δ 148.62 (C), 141.81 (CH), 138.57 (C), 132.72 (CH),130.49 (CH), 91.51 (C), 73.48 (CH), 34.22 (CH), 19.62 (CH₃), 16.71(CH₃).

(R)- and (S)-1-(4-iodo-2-nitrophenyl)-2-methyl-propyl (1S)-camphanate:Under a nitrogen atmosphere (1S)-camphanic acid chloride (0.89 g, 4.09mmol) was added to a solution of(RS)-1-(4-Iodo-2-nitrophenyl)-2-methyl-1-propanol (1.10 g, 3.41 mmol)and DMAP (0.50 g, 4.09 mmol) in anhydrous dichloromethane (30 mL). Themixture was stirred for 1.5 hours at room temperature and thenconcentrated in vacuo. The residue was purified by silica gel columnchromatography to (RS)-1-(4-Iodo-2-nitrophenyl)-2-methyl-1-propyl(1S)-camphanates (1.44 g, 84%, 1:1 mixture of diastereomers) as a solid,which was dissolved in boiling methanol (70 mL) and left at roomtemperature overnight. Crystals formed were filtered to yield pure(R)-1-(4-iodo-2-nitrophenyl)-2-methyl-1-propyl (1S)-camphanate (0.465 g,65%). The mother liquor was evaporated, and the residue was dissolved inboiling isopropanol (40 mL) and left at room temperature overnight.Crystals formed were filtered and recrystallized from boilingisopropanol (20 mL) to yield pure(S)-1-(4-iodo-2-nitrophenyl)-2-methyl-1-propyl (1S)-camphanate (0.288 g,40%). ¹H NMR (400 MHz, CDCl₃) for (R,S)-camphanate: δ 8.30 (d, 1 H,J=1.8 Hz, Ph-H), 7.92 (dd, 1 H, J=1.8 and 8.3 Hz, Ph-H), 7.56 (d, 1 H,J=8.3 Hz, Ph-H), 6.27 (d, 1 H, J=6.8 Hz, Ph-CH), 2.40 (m, 1 H), 2.23 (m,1 H), 2.06 (m, 1 H), 1.92 (m, 1 H), 1.72 (m, 1 H), 1.11 (s, 3 H, CH₃),1.03 (d, J=6.8 Hz, 3 H, CH₃), 1.02 (s, 1 H, CH₃), 0.98 (d, J=6.8 Hz, 3H, CH₃), 0.82 (s, 3 H, CH₃); ¹³C NMR (100 MHz, CDCl₃) for(R,S)-camphanate: δ 178.21 (C), 166.92 (C), 148.67 (C), 142.07 (CH),134.80 (C), 133.34 (CH), 129.73 (CH), 92.59 (C), 90.75 (C), 76.07 (CH),54.79 (C), 54.34 (C), 33.27 (CH), 31.03 (CH₂), 28.88 (CH₂), 19.07 (CH₃),17.40 (CH₃), 16.70 (CH₃), 16.66 (CH₃), 9.61 (CH₃).

¹H NMR (400 MHz, CDCl₃)) for (S,S)-camphanate: δ 8.30 (d, 1 H, J=1.8 Hz,Ph-H), 7.94 (dd, 1 H, J=1.8 and 8.3 Hz, Ph-H), 7.35 (d, 1 H, J=8.3 Hz,Ph-H), 6.23 (d, 1 H, J=6.1 Hz, Ph-CH), 2.34 (m, 1 H), 2.24 (m, 1 H),1.91 (m, 2 H), 1.67 (m, 1 H), 1.13 (s, 3 H, CH₃), 1.04 (s, 3 H, CH₃),1.02 (d, 3 H, J=5.2 Hz, CH₃), 1.00 (s, 3 H, CH₃), 0.98 (d, 3 H, J=5.6Hz, CH₃); ¹³C NMR (100 MHz, CDCl₃) (S,S)-camphanate: δ 178.28 (C),167.03 (C), 148.67 (C), 142.27 (CH), 134.90 (C), 133.28 (CH), 129.59(CH), 91.60 (C), 91.06 (C), 76.21 (CH), 54.91 (C), 54.40 (C), 33.16(CH), 30.75 (CH₂), 28.83 (CH₂), 19.14 (CH₃), 17.41 (CH₃), 16.97 (CH₃),16.65 (CH₃), 9.70 (CH₃).

X-ray crystallography data for(R)-1-(4-iodo-2-nitrophenyl)-2-methyl-1-propyl (1S)-camphanate: Crystaldata for lg₀₁a: C₂₀H₂₄INO₆, M=501.30, colorless plate, 0.30×0.20×0.20mm³, monoclinic, space group P2₁ (No. 4), a=7.5810(15), b=12.446(3),c=11.722(3) Å, β=107.613(10)°, V=1054.2(4) Å³, Z=2, D_(c)=1.579 g/cm³,F₀₀₀=504, CCD area detector, MoKα radiation, λ=0.71073 Å, T=110(2)K,2θ_(max)=50.0°, 24239 reflections collected, 3558 unique(R_(int)=0.0302). Final GooF=1.010, R1=0.0123, wR2=0.0316, R indicesbased on 3520 reflections with I>2sigma(I) (refinement on F²), 253parameters, 3 restraints. Lp and absorption corrections applied, μ=1.554mm¹. Absolute structure parameter=0.020(9) (Flack, 1983, which isincorporated herein by reference). See FIG. 3.

(R)-1-(4-Iodo-2-nitrophenyl)-2-methyl-1-propanol:(R)-1-(4-Iodo-2-nitrophenyl)-2-methyl-1-propyl (1S)-camphanate (0.41 g,0.82 mmol) was dissolved in hot methanol (60 mL) and K₂CO₃ (0.22 g, 1.58mmol) was added. The mixture was heated to reflux for one hour, cooledto room temperature and concentrated in vacuo. The residue was purifiedby silica gel column chromatography to yield(R)-1-(4-Iodo-2-nitrophenyl)-2-methyl-1-propanol (0.262 g, 100%) as alight yellow oil. ¹H NMR was identical to that of the racemic alcohol.

(S)-1-(4-Iodo-2-nitrophenyl)-2-methyl-1-propanol:(S)-1-(4-Iodo-2-nitrophenyl)-2-methyl-1-propyl (1S)-camphanate (0.288 g,0.57 mmol) was dissolved in hot methanol (40 mL) and K₂CO₃ (0.15 g, 1.1mmol) was added. The mixture was heated to reflux for one hour, cooledto room temperature and concentrated in vacuo. The residue was purifiedby silica gel column chromatography to yield(S)-1-(4-Iodo-2-nitrophenyl)-2-methyl-1-propanol (0.184 g, 100%) as alight yellow oil. ¹H NMR was identical to that of the racemic alcohol.

Synthesis of (RS)-1-(2-nitrophenyl)-2,2-dimethyl-1-propanol and (R orS)-1-(2-nitrophenyl)-2,2-dimethyl-1-propanol

(RS)-1-(2-Nitrophenyl)-2,2-dimethyl-1-propanol: Under a nitrogenatmosphere, a solution of 1-iodo-2-nitrobenzene (3.0 g, 12 mmol) inanhydrous THF (30 mL) was cooled to minus 40° C., and thenphenylmagnesium chloride (2 M in THF, 7.2 mL, 14.5 mmol) was addeddropwise at a rate that the temperature would not exceed minus 35° C.After the mixture was stirred for 20 minutes at minus 40° C.,trimethylacetaldehyde (1.85 mL, 16.8 mmol) was added dropwise and themixture was stirred for another 30 minutes at minus 40° C. The mixturewas gradually warmed up to room temperature, quenched with saturatedammonium chloride (60 mL), poured into water (60 mL), and extracted withethyl acetate three times (60 mL each). The combined organic phase wasdried over Na₂SO₄, and concentrated in vacuo. The residue was purifiedby silica gel column chromatography to yield(RS)-1-(2-nitrophenyl)-2,2-dimethyl-1-propanol (1.82 g, 72%) as a yellowsolid. ¹H NMR (400 MHz, CDCl₃): δ 7.82 (d, 1 H, J=6.4 Hz, Ph-H), 7.76(d, 1 H, J=6.4 Hz, Ph-H), 7.61 (t, 1 H, J=6.4 Hz, Ph-H), 7.39 (t, 1 H,J=6.4 Hz, Ph-H), 5.39 (d, 1 H, J=2.8 Hz, Ph-CH), 2.14 (d, 1 H, J=3.2 Hz,OH), 0.89 (s, 9 H, C(CH₃)₃).

(RS)-1-(2-Nitrophenyl)-2,2-dimethyl-1-propyl (1S)-camphanate: Under anitrogen atmosphere, (1S)-camphanic acid chloride (3.37 g, 15.6 mmol)was added to a solution of(RS)-1-(2-nitrophenyl)-2,2-dimethyl-1-propanol (2.71 g, 13 mmol) andDMAP (80 mg, 0.65 mmol) in anhydrous pyridine (50 mL). The mixture wasstirred for 24 hours at room temperature. Solvent was removed in vacuo,and the residue was dissolved in ethyl acetate (50 mL), washed with 0.5M HCl (20 mL) twice followed with saturated NaHCO₃ solution (20 mL). Theorganic phase was dried over Na₂SO₄, concentrated in vacuo and purifiedby silica gel column chromatography to yield(RS)-1-(2-nitrophenyl)-2,2-dimethyl-1-propyl (1S)-camphanate (4.11 g,81%, 1:1 mixture of diastereomers) as a yellow solid. ¹H NMR (400 MHz,CDCl₃) for diastereomers: δ 7.91 (m, 1 H, Ph-H), 7.62 (m, 2 H, Ph-H),7.46 (m, 1 H, Ph-H), 6.66 and 6.62 (2 s, 1 H, Ph-CH), 2.38 (m, 1 H),2.10-1.9 (m, 2 H), 1.71 (m, 1 H), 1.13, 1.11, 1.08, 1.04, 1.03, 0.87 (6s, 9 H, CH₃×3), 0.97 (s, 9 H, (CH₃)₃C).

(R or S)-1-(2-Nitrophenyl)-2,2-dimethyl-1-propyl (1S)-amphanate: Puresingle diastereomer (R or S)-1-(2-nitrophenyl)-2,2-dimethyl-1-propyl(1S)-camphanate (0.79 g, 35%) was obtained after repeatedcrystallization of (RS)-1-(2-Nitrophenyl)-2,2-dimethyl-1-propyl(1S)-camphanate (4.57 g) from methanol. The absolute configuration ofthe single diastereomer camphanate is not determined. ¹H NMR (400 MHz,CDCl₃): δ 7.91 (dd, 1 H, J=0.8 and 8.0 Hz, Ph-H), 7.61 (m, 2 H, Ph-H),7.46 (m, 1 H, Ph-H), 6.62 (s, 1 H, Ph-CH), 2.35 (m, 1 H), 1.93 (m, 2 H),1.69 (m, 1 H), 1.13, 1.08 and 1.02 (3 s, 9 H, CH₃×3), 0.96 (s, 9 H,(CH₃)₃C).

(R or S)-1-(2-Nitrophenyl)-2,2-dimethyl-1-propanol: A mixture of singlediastereomer (R or S)-1-(2-nitrophenyl)-2,2-dimethyl-1-propyl(1S)-camphanate (658 mg, 1.68 mmol) and K₂CO₃ (241 mg, 1.74 mmol) inmethanol (23 mL) was heated to reflux for 30 minutes. Water (5 mL) wasadded and the solution was neutralized to pH 7 with 1 M HCl. Solvent wasremoved in vacuo and the residue was taken into a mixture of ethylacetate (20 mL) and water (10 mL). The organic phase was separated,dried over anhydrous Na₂SO₄, concentrated in vacuo and purified bysilica gel column chromatography to yield single enantiomer (R orS)-1-(2-nitrophenyl)-2,2-dimethyl-1-propanol (325 mg, 92%) as a lightyellow oil. ¹H NMR was identical to that of the racemic alcohol. Theabsolute configuration of the single enantiomer alcohol is notdetermined.

Synthesis of (RS)-1-(4-Iodo-2-nitrophenyl)-2,2-dimethyl-1-propanol and(R or S)-1-(4-Iodo-2-nitrophenyl)-2,2-dimethyl-1-propanol

(RS)-1-(4-Iodo-2-nitrophenyl)-2,2-dimethyl-1-propanol: Under a nitrogenatmosphere a solution of 1,4-diiodo-2-nitrobenzene (3.0 g, 8.0 mmol) inanhydrous THF (20 mL) was cooled to minus 40° C., and then a solution ofphenylmagnesium chloride (2 M in THF, 4.8 mL, 9.6 mmol) was addeddropwise at a rate that the temperature would not exceed minus 35° C.Upon completion of the addition the mixture was stirred for ten minutes,followed by addition of trimethylacetaldehyde (1.2 mL, 11.2 mmol), andthe mixture was stirred for 30 minutes at minus 40° C. The mixture wasgradually warmed up to room temperature, quenched with saturatedammonium chloride (60 mL), poured into water (120 mL), and extractedwith ethyl acetate twice (60 mL each). The combined organic phase waswashed with water (60 mL), dried over Na₂SO₄, and concentrated in vacuo.The residue was purified by silica gel column chromatography to yieldracemic (RS)-1-(4-iodo-2-nitrophenyl)-2,2-dimethyl-1-propanol (2.17 g,81%) as a brown oil. ¹H NMR (400 MHz, CDCl₃): δ 8.04 (d, 1 H, J=1.6 Hz,Ph-H), 7.88 (dd, 1 H, J=1.6 and 8.4 Hz, Ph-H), 7.51 (d, 1 H, J=8.4 Hz,Ph-H), 5.28 (d, 1 H, J=3.6 Hz, Ph-CH), 2.29 (d, 1 H, J=3.6 Hz, OH), 0.85(s, 9 H, C(CH₃)₃). ¹³C NMR (100 MHz, CDCl₃): δ 149.87 (C), 141.0 (CH),136.2 (C), 132.3 (CH), 131.63 (CH), 91.85 (C), 74.33 (CH), 36.81 (C),25.6 (CH₃).

(RS)-1-(4-Iodo-2-nitrophenyl)-2,2-dimethyl-1-propyl (1S)-camphanates:Under a nitrogen atmosphere, (1S)-camphanic acid chloride (1.68 g, 7.77mmol) was added to a solution of(RS)-1-(4-iodo-2-nitrophenyl)-2,2-dimethyl-1-propanol (2.17 g, 6.47mmol) and DMAP (1.18 g, 9.7 mmol) in anhydrous dichloromethane (50 mL).The mixture was stirred overnight at room temperature and then washedwith saturated NaHCO₃ solution (60 mL) and water (60 mL). The organicphase was dried over Na₂SO₄, concentrated in vacuo and purified bysilica gel column chromatography to yield(RS)-1-(4-iodo-2-nitrophenyl)-2,2-dimethyl-1-propyl (1S)-camphanate(2.94 g, 88%, 1:1 mixture of diastereomers) as a white solid. ¹H NMR(400 MHz, CDCl₃) for diastereomers: δ 8.23 (m, 1 H, Ph-H), 7.90 (m, 1 H,Ph-H), 7.31 (m, 1 H, Ph-H), 6.56 and 6.51 (2 s, 1 H, Ph-CH), 2.38 (m, 1H), 2.07-1.9 (m, 2 H), 1.69 (m, 1 H), 1.13, 1.11, 1.07, 1.04, 1.02, 0.87(6 s, 9 H, CH₃×3), 0.96 (s, 9 H, (CH₃)₃C).

(R or S)-1-(4-Iodo-2-nitrophenyl)-2,2-dimethyl-1-propyl (1S)-camphanate:(RS)-1-(4-Iodo-2-nitrophenyl)-2,2-dimethyl-1-propyl (1S)-camphanate(2.07 g) was dissolved in boiling methanol (100 mL) and left at roomtemperature for two days. Crystals formed were filtered to yield puresingle diastereomer (R or S)-1-(4-iodo-2-nitrophenyl)-2-methyl-1-propyl(1S)-camphanate (0.409 g, 39%). The absolute configuration of the singlediastereomer camphanate is not determined. ¹H NMR (400 MHz, CDCl₃): δ8.23 (d, 1 H, J=2.0 Hz, Ph-H), 7.89 (dd, 2 H, J=2.0 and 8.4 Hz, Ph-H),7.30 (d, 1 H, J=8.4 Hz, Ph-H), 6.56 (s, 1 H, Ph-CH), 2.42 (m, 1 H), 2.07(m, 1 H), 1.94 (m, 1 H), 1.73 (m, 1 H), 1.11, 1.04 and 0.87 (3 s, 9 H,CH₃ 3), 0.95 (s, 9 H, (CH₃)₃C).

(R or S)-1-(4-Iodo-2-nitrophenyl)-2,2-dimethyl-1-propanol: A mixture ofsingle diastereomer (R orS)-1-(4-iodo-2-nitrophenyl)-2,2-dimethyl-1-propyl (1S)-camphanate (409mg, 0.79 mmol) and K₂CO₃ (110 mg, 0.8 mmol) in methanol (15 mL) washeated to reflux for 30 minutes. Water (5 mL) was added and the solutionwas neutralized to pH 7 with 1 M HCl. Solvent was removed in vacuo andthe residue was taken into a mixture of ethyl acetate (30 mL) and water(10 mL). The organic phase was separated, dried over anhydrous Na₂SO₄,concentrated in vacuo, and purified by silica gel column chromatographyto yield single enantiomer (R orS)-1-(4-iodo-2-nitrophenyl)-2,2-dimethyl-1-propanol (260 mg, 98%) as alight yellow oil. ¹H NMR was identical to that of the racemic alcohol.The absolute configuration of the single enantiomer alcohol is notdetermined.

Synthesis of (±)-1-(2,6-dinitrophenyl)-2-methyl-1-propanol

1-Iodo-2,2-dinitrobenzene: To a dispersion of 2,6-dinitroaniline (4.00g, 0.021 mol) in diiodomethane (24 mL, 0.297 mol) isoamylnitrite (15 mL,0.112 mol) was added (Smith et al., 1990, which is incorporated hereinby reference). The mixture was stirred at room temperature for one hour,and then heated at 105° C. for eight hours. The excess of diiodomethanewas removed in high vacuo. The residue was diluted with acetyl acetate(10 mL). Silica (ca 30 mL, mesh 230-400 Å) was added; the mixture wasconcentrated in vacuo and purified by column chromatography to yield1-iodo-2,6-dinitrobenzene (3.21 g, 52%). ¹H NMR (400 MHz, CDCl₃): δ 7.84(d, 2 H, J=8.0 Hz), 7.67 (t, 1 H, J=8.0 Hz).

(±)-1-(2,2-dinitrophenyl)-2-methyl-1-propanol: To a solution of1-iodo-2,6-dinitrobenzene (1.55 g, 5.27 mmol) in anhydroustetrahydrofuran (18 mL) cooled at minus 53° C. (dry ice-isopropanolbath) under nitrogen atmosphere, phenylmagnesium bromide (2 M in THF,3.16 mL, 6.32 mmol) was added at a rate to keep the temperature at orbelow minus 45° C. Upon completion of the addition the mixture wasstirred for five minutes, then isobutyraldehyde (0.957 mL, 10.54 mmol)was added. The mixture was allowed to gradually warm up to roomtemperature, then quenched with saturated NH₄Cl (5 mL) and poured intowater (50 mL)/dichloromethane (100 mL). The organic layer was separated;aqueous layer was extracted three times with dichloromethane (50 mLeach). Combined organic extract was washed with water (20 mL), driedover anhydrous Na₂SO₄, concentrated under reduced pressure, and purifiedby silica gel column chromatography to yield racemic(RS)-1-(2,6-dinitrophenyl)-2-methyl-1-propanol (0.375 g, 30%) as a lightyellow oil. ¹H NMR (400 MHz, CDCl₃): δ 7.82 (d, 2 H, J=8.0 Hz, Ph-H),7.59 (t, 1 H, J=8.0 Hz, Ph-H), 4.84 (dd, 1 H, J=7.6 and 9.2 Hz, Ph-CH),2.87 (d, 1 H, J=7.6 Hz, OH), 2.18 (m, 1 H, CHCH(CH₃)₂), 1.12 (d, 3 H,J=6.4 Hz, CH₃), 0.77 (d, 3 H, J=6.8 Hz, CH₃); ¹³C NMR (100 MHz, CDCl₃):δ 150.82 (C), 130.62 (C), 129.31 (CH), 127.30 (CH), 74.52 (CH), 34.19(CH), 19.84 (CH₃), 19.14 (CH₃).

Synthesis of (±)-1-(4-methoxy-2-nitrophenyl)-2-methyl-1-propanol

(±)-1-(4-Methoxy-2-nitrophenyl)-2-methyl-1-propanol: To a solution of4-iodo-2-nitroanisole (2.79 g, 10.00 mmol) in anhydrous tetrahydrofuran(20 mL) cooled at minus 45° C. (dry ice-isopropanol bath) under nitrogenatmosphere, phenylmagnesium chloride (2 M in THF, 6 mL, 6.32 mmol) wasadded at a rate to keep the temperature at or below minus 40° C. Uponcompletion of the addition the mixture was stirred for five minutes,then isobutyraldehyde (1.816 mL, 20.00 mmol) was added. The mixture wasallowed to gradually warm up to room temperature, then quenched withsaturated NH₄Cl (5 mL) and poured into water (30 mL)/dichloromethane(100 mL). The organic layer was separated; aqueous layer was extractedthree times with dichloromethane (50 mL each). Combined organic extractwas dried over anhydrous Na₂SO₄, concentrated under reduced pressure,and purified by silica gel column chromatography to yield racemic(RS)-1-(4-methoxy-2-nitrophenyl)-2-methyl-1-propanol (1.502 g, 30%) as ayellow oil. ¹H NMR (400 MHz, CDCl₃): δ 7.62 (AB d, 1 H, J=8.8 Hz, Ph-H),7.34 (d, 1 H, J=2.6 Hz, Ph-H), 7.15 (dd, 1 H, J=2.6 and 8.8 Hz, Ph-H),4.91 (m, 1 H, Ph-CH), 3.86 (s, 3H, MeO), 2.45 (br s, 1 H, OH), 2.00 (m,1 H, CHCH, (CH₃)₂), 0.97 (d, 3 H, J=6.7 Hz, CH₃), 0.86 (d, 3 H, J=6.9Hz, CH₃); ¹³C NMR (100 MHz, CDCl₃): δ 158.76 (C), 149.07 (C), 130.79(C), 129.88 (CH), 119.62 (CH), 108.66 (CH), 73.75 (CH), 55.81 (CH₃),34.27 (CH), 19.59 (CH₃), 17.36 (CH₃).

Synthesis (±)-1-(5-cyano-2-nitrophenyl)-2-methyl-1-propanol

3-iodo-4-nitrobenzonitrile: To a suspension of 4-amino-3-iodobenzene(2.44 g, 10.00 mmol) in aqueous sulfuric acid (2 M, 50 mL) chilled to 0°C. (ice-water-NaCl bath) a solution of sodium nitrite (1.656 g, 24.00mol) in water (6 mL) was added at such rate that the temperature of thereaction mixture did not exceed 0° C. Upon completion of the additionthe mixture was stirred for one hour (or until cleared), then it wastransferred in portions to a hot solution of sodium nitrite (13.8 g, 200mmol) and CuSO₄.5H₂O (0.12 g, 0.05 mmol). (CAUTION: vigorous gasevolution!) Upon completion of transfer, the mixture was stirred atreflux for 30 minutes, then cooled down to room temperature, andextracted three times with dichloromethane (100 mL each). Combinedextracts were dried over Na₂SO₄, concentrated under reduced pressure,mixed with silica (ca 30 mL, mesh 230-400 Å), and purified by columnchromatography to yield 3-iodo-4-nitrobenzonitrile (0.95 g, 35%). ¹H NMR(400 MHz, CDCl₃): δ 8.10 (d, 1 H, J=1.9 Hz), 7.99 (d, 1 H, J=8.2 Hz),7.29 (dd, 1 H, 8.2 and 1.9 Hz, 1 H); ¹³C NMR (100 MHz, CDCl₃): δ 141.76(CH), 139.93 (CH), 131.66 (CH), 116.65 (C), 114.78 (CN), 113.15 (C),108.62 (C).

(±)-1-(5-cyano-2-nitrophenyl)-2-methyl-1-propanol: To a solution of3-iodo-4-nitrobenzonitrile (314 mg, 1.14 mmol) in anhydroustetrahydrofuran (5.5 mL) cooled at minus 45° C. (dry ice-isopropanolbath) under nitrogen atmosphere, phenylmagnesium bromide (2 M in THF,3.16 mL, 6.32 mmol) was added at a rate to keep the temperature at orbelow minus 40° C. Upon completion of the addition the mixture wasstirred for five minutes, then isobutyraldehyde (0.957 mL, 10.54 mmol)was added. The mixture was allowed to gradually warm up to roomtemperature, then quenched with saturated NH₄Cl (2 mL) and poured intowater (50 mL)/dichloromethane (50 mL). The organic layer was separated;aqueous layer was extracted three times with dichloromethane (25 mLeach). Combined organic extract was dried over anhydrous Na₂SO₄,concentrated under reduced pressure, and purified by silica gel columnchromatography to yield racemic(RS)-1-(5-cyano-2-nitrophenyl)-2-methyl-1-propanol (57 mg, 23%) as alight yellow oil. ¹H NMR (400 MHz, CDCl₃): δ 7.93 (d, 1 H, J=8.1 Hz,Ph-H), 7.74 (s, 1 H, Ph-H), 7.21 (d, 1 H, J=8.1 Hz, Ph-H), 4.72 (d, 1 H,J=3.6 Hz, Ph-CH), 2.33 (br. s, 1 H, OH), 2.03 (m, 1 H, CHCH(CH₃)₂), 1.04(d, 3 H, J=6.8 Hz, CH₃), 0.89 (d, 3 H, J=6.6 Hz, CH₃); ¹³C NMR (100 MHz,CDCl₃): δ 147.78 (C), 140.25 (CH), 131.40 (CH), 131.24 (CH), 118.36 (C),112.27 (CN), 103.82 (C), 80.80 (CH), 33.56 (CH), 19.70 (CH₃), 15.62(CH₃).

Example 3 Synthesis of Deoxyuridine and Deoxycytidine Analogs withα-Isopropyl Groups Synthesis5-[(S)-1-(2-nitrophenyl)-2-methyl-propyloxy]methyl-2′-deoxyuridine-5′-triphosphate

5-[(S)-1-(2-Nitrophenyl)-2-methyl-propyloxy]methyl-2′-deoxyuridine(dU.x1): Compound dU.x0 (250 mg, 0.385 mmol) and(S)-1-(2-nitrophenyl)-2-methyl-propanol (300 mg, 1.54 mmol) were heatedneat at 105-110° C. for 30 minutes under a nitrogen atmosphere. Themixture was cooled down to room temperature, dissolved in minimum amountof ethyl acetate, and purified by silica gel chromatography to yield5-[(S)-1-(2-nitrophenyl)-2-methyl-propyloxy]methyl-2′-deoxyuridine dU.x1(6 mg, 4%). (3′ or5)-O-(tert-butyldimethylsilyl)-5-[(S)-1-(2-nitrophenyl)-2-methyl-propyloxy]methyl-2′-deoxyuridine(39 mg, 18%) and3′,5′-O-bis-(tert-butyldimethylsilyl)-5-[(S)-1-(2-nitrophenyl)-2-methyl-propyloxy]methyl-2′-deoxyuridine(36 mg, 14%) were also obtained from the reaction. ¹H NMR (100 MHz,CD₃OD) for dU.x1: δ 7.96 (s, 1 H, H-6), 7.88 (d, 1 H, J=8.3 Hz, Ph-H),7.74 (dd, 1 H, J=1.5 and 7.6 Hz, Ph-H), 7.68 (m, 1 H, Ph-H), 7.49 (m, 1H, Ph-H), 6.25 (dd, 1 H, J=6.4 and 7.8 Hz, H-1′), 4.77 (d, 1 H, J=6.0Hz, Ph-CH), 4.39 (m, 1 H, H-3′), 4.17 (AB d, 1 H, J=12.3 Hz, 5-CH₂a),4.07 (AB d, 1 H, J=12.3 Hz, 5-CH₂b), 3.92 (m, 1 H, H-4′), 3.77 (AB dd, 1H, J=3.4 and 12.1 Hz, H-5′a), 3.71 (AB dd, 1 H, J=3.8 and 12.1 Hz,H-5′b), 2.24 (m, 2 H, H-2′), 1.95 (m, 1 H, CH), 0.94 (d, 3 H, J=6.7 Hz,CH₃), 0.85 (d, 3 H, J=6.9 Hz, CH₃).

5-[(S)-1-(2-Nitrophenyl)-2-methyl-propyloxy]methyl-2′-deoxyuridine-5′-triphosphate(WW3p063): POCl₃ (4 μL, 0.045 mmol) was added to a solution of compounddU.x1 (14 mg, 0.03 mmol) and proton sponge (13 mg, 0.06 mmol) intrimethylphosphate (0.3 mL) at 0° C. and stirred for one hour.Additional POCl₃ (4 μL, 0.045 mmol) was added and the mixture wasstirred for another three hours. A solution of tri-n-butylammoniumpyrophosphate (72 mg, 0.15 mmol) and tri-n-butylamine (30 μL) inanhydrous DMF (0.3 mL) was added. After five minutes of stirring,triethylammonium bicarbonate buffer (1 M, pH 7.5; 5 mL) was added. Thereaction was stirred at room temperature for one hour and thenlyophilized to dryness. The residue was dissolved in water (5 mL),filtered, and purified by anion exchange chromatography on a Q SepharoseFF column (2.5×20 cm) with a linear gradient of NH₄HCO₃ (50 mM to 500 mMin 240 minutes) at a flow rate of 4.5 mL/min. The fractions containingtriphosphate were combined and lyophilized to give5-[(S)-1-(2-nitrophenyl)-2-methyl-propyloxy]methyl-2′-deoxyuridine-5′-triphosphateWW3p063 (10 mg, 46%) as a white fluffy solid. ¹H NMR (400 MHz, D₂O): δ7.88 (dd, 1 H, J=1.2 and 6.4 Hz, Ph-H), 7.66 (m, 1 H, Ph-H), 7.58 (dd, 1H, J=1.2 and 6.4 Hz, Ph-H), 7.56 (s, 1 H, H-6), 7.46 (dt, 1 H, J=1.2 and6.4 Hz, Ph-H), 6.09 (t, 1 H, J=5.6 Hz, H-1′), 4.46 (m, 1 H, H-3′), 4.39(AB d, 1 H, J=10 Hz, 5-CH₂a), 4.23 (AB d, 1 H, J=10 Hz, 5-CH₂b),4.20-4.12 (m, 3 H, H-4′ and H-5′), 2.29 (m, 1 H, H-2′a), 2.2 (m, 1 H,H-2′b), 1.94 (m, 1 H, CH), 0.97 (d, 3 H, J=5.6 Hz, CH₃), 0.74 (d, 3 H,J=5.6 Hz, CH₃). ³¹P NMR (162 MHz, D₂O): δ −5.06 (d, J=16.2 Hz), −10.55(d, J=16.2 Hz), −20.9 (t, J=16.2 Hz).

Synthesis5-[(S)-1-(2-nitrophenyl)-2-methyl-propyloxy]methyl-2′-deoxycytidine-5′-triphosphate

3′,5′-O-Bis-(tert-butyldimethylsilyl)-5-[(S)-1-(2-nitrophenyl)-2-methyl-propyloxy]methyl-2′-deoxyuridine(dU.x2): Compound dU.x0 (250 mg, 0.385 mmol) and(S)-1-(2-nitrophenyl)-2-methyl-propanol (300 mg, 1.54 mmol) were heatedneat at 105-110° C. for 30 minutes under a nitrogen atmosphere. Themixture was cooled down to room temperature, dissolved in minimum amountof ethyl acetate, and purified by silica gel chromatography to yield3′,5′-O-bis-(tert-butyldimethylsilyl)-5-[(S)-1-(2-nitrophenyl)-2-methyl-propyloxy]methyl-2′-deoxyuridinedU.x2 (36 mg, 14%). (3′ or5′)—O-(tert-butyldimethylsilyl)-5-[(S)-1-(2-nitrophenyl)-2-methyl-propyloxy]methyl-2′-deoxyuridine(39 mg, 18%) and5-[(S)-1-(2-nitrophenyl)-2-methyl-propyloxy]methyl-2′-deoxyuridine (6mg, 4%) were also obtained from the reaction. ¹H NMR (400 MHz, CDCl₃)for dU.x2: δ 8.76 (s, 1 H, NH), 7.84 (dd, 1 H, J=1.1 and 8.1 Hz, Ph-H),7.73 (dd, 1 H, J=1.3 and 7.9 Hz, Ph-H), 7.66 (m, 1 H, Ph-H), 7.59 (s, 1H, H-6), 7.42 (m, 1 H, Ph-H), 6.29 (dd, 1 H, J=5.9 and 7.8 Hz, H-1′),4.78 (d, 1 H, J=6.2 Hz, Ph-CH), 4.42 (m, 1 H, H-3′), 4.18 (AB d, 1 H,J=12.0 Hz, 5-CH₂a), 4.04 (AB d, 1 H, J=12.0 Hz, 5-CH₂b), 3.94 (m, 1 H,H-4′), 3.77 (m, 2 H, H-5′), 2.30 (m, 1 H, H-2′a), 2.05 (m, 1 H, H-2′b),1.96 (m, 1 H, CH), 0.92 (d, 3 H, J=6.7 Hz, CH₃), 0.90 (s, 9 H,(CH₃)₃CSi), 0.89 (s, 9 H, (CH₃)₃CSi), 0.84 (d, 3 H, J=6.9 Hz, CH₃), 0.10(s, 3 H, CH₃Si), 0.09 (s, 3 H, CH₃Si), 0.08 (s, 3 H, CH₃Si), 0.07 (s, 3H, CH₃Si); ¹³C NMR (100 MHz, CDCl₃) for dU.x2: δ 162.39 (C), 150.01 (C),149.47 (C), 138.33 (CH), 136.78 (C), 132.88 (CH), 129.24 (CH), 124.08(CH), 111.48 (C), 87.84 (CH), 85.16 (CH), 81.33 (CH), 72.35 (CH), 64.50(CH₂), 63.09 (CH₂), 40.95 (CH₂), 34.91 (CH), 25.94 (C(CH₃)₃), 25.76(C(CH₃)₃), 19.28 (CH₃), 18.41 (C), 18.02 (C), 17.90 (CH₃), −4.66 (CH₃),−4.82 (CH₃), −5.32 (CH₃), −5.41 (CH₃).

3′,5′-O-Bis-(tert-butyldimethylsilyl)-5-[(S)-1-(2-nitrophenyl)-2-methyl-propyloxy]methyl-O⁴-(2,4,6-triisopropylbenzenesulfonyl)-2′-deoxyuridine(dU.x3):To a solution of compound dU.x2 (90 mg, 0.136 mmol), DMAP (17mg, 0.140 mmol), and triethylamine (0.172 mL, 1.224 mmol) in anhydrousdichloromethane (6 mL) 2,4,6-triisopropylbenzenesulfonyl chloride (1.57g, 5.19 mmol) was added. The mixture was stirred at room temperature for17 hours under nitrogen atmosphere, then concentrated in vacuo andpurified by column chromatography to afford3′,5′-O-bis-(tert-butyldimethylsilyl)-5-[(S)-1-(2-nitrophenyl)-2-methyl-propyloxy]methyl-4-O-(2,4,6-triisopropylbenzenesulfonyl)-2′-deoxyuridinedU.x3 (54 mg, 42%). ¹H NMR (400 MHz, CDCl₃): δ 8.07 (s, 1 H, H-6), 7.86(m, 1 H, Ph-H), 7.71 (m, 1 H, Ph-H), 7.46 (m, 1 H, Ph-H), 7.16 (s, 2 H,Ph-H), 6.09 (t, 1 H, J=6.4 Hz, H-1′), 4.79 (d, 1 H, J=6.2 Hz, Ph-CH),4.34 (m, 1 H, H-3′), 4.12 (m, 4 H), 3.97 (m, 1 H, H-4′), 3.80 (AB dd, 1H, J=3.4 and 11.2 Hz, H-5′a), 3.80 (AB dd, 1 H, J=3.6 and 11.2 Hz,H-5′b), 2.89 (m, 1 H, CH), 2.51 (m, 1 H, H-2′a), 1.96 (m, 2 H), 1.29 and1.21 (d, 12 H, J=6.7 Hz, CH₃×4), 1.25 (d, 6 H, J=7.0 Hz, CH₃×2), 0.99(d, 3 H, J=6.7 Hz, CH₃), 0.87 (2 s, 18 H, (CH₃)₃CSi), 0.84 (d, 3 H,J=6.9 Hz, CH₃), 0.07 (s, 6 H, (CH₃)₂Si), 0.06 (s, 6 H, (CH₃)₂Si).

3′,5′-O-Bis-(tert-butyldimethylsilyl)-5-[(S)-1-(2-nitrophenyl)-2-methyl-propoxy]methyl-2′-deoxcytidine(dC.x4): To a solution of compound dU.x3 (47 mg, 0.051 mmol) inanhydrous 1,4-dioxane (3 mL) a solution of ammonia (3 mL, 0.5 M indioxane, 1.50 mmol) was added. The mixture was transferred into a sealedtube and heated at 85-90° C. for 1.5 hours. The mixture was cooled downto room temperature, concentrated in vacuo and purified by silica gelcolumn chromatography to yield3′,5′-O-bis-(tert-butyldimethylsilyl)-5-[(S)-1-(2-nitrophenyl)-2-methyl-propoxy]methyl-2′-deoxcytidinedC.x4 (20 mg, 61%) as a waxy solid. ¹H NMR (400 MHz, CDCl₃) δ 7.81 (d, 1H, J=8.1 Hz, Ph-H), 7.66 (m, 2 H, Ph-H), 7.51 (s, 1 H, H-6), 7.45 (m, 1H, Ph-H), 6.64 and 5.81 (2 br. s, 2 H, NH₂), 6.28 (t, 1 H, J=6.5 Hz,H-1′), 4.70 (d, 1 H, J=6.8 Hz, Ph-CH), 4.32 (m, 1 H, H-3′), 4.20 (AB d,1 H, J=12.6 Hz, 5-CH₂a), 4.07 (AB d, 1 H, J=12.6 Hz, 5-CH₂b), 3.89 (m, 1H, H-4′), 3.77 (AB dd, 1H, J=3.5 and 11.2 Hz, H-5′a), 3.69 (AB dd, 1 H,J=3.4 and 11.2 Hz, H-5′b), 2.42 (m, 1 H, H-2′a), 1.99 (m, 2 H, H-2′b andCH), 0.98 (d, 3 H, J=6.6 Hz, CH₃), 0.88 (s, 9 H, (CH₃)₃CSi), 0.80 (s, 9H, (CH₃)₃CSi), 0.78 (d, 3 H, J=7.0 Hz, CH₃), 0.07 (s, 3 H, CH₃Si), 0.06(s, 3 H, CH₃Si), −0.01 (s, 3 H, CH₃Si), −0.04 (s, 3 H, CH₃Si).

5-[(S)-1-(2-nitrophenyl)-2-methyl-propyloxy]methyl-2′-deoxcytidine(dC.x5): To a solution of compound dC.x4 (16 mg, 0.024 mmol) in THF (1mL) a solution of tetra-n-butylammonium fluoride trihydrate (31 mg,0.096 mmol) in THF (2 mL) was added. The mixture was stirred at roomtemperature for 30 minutes, concentrated in vacuo and purified by silicagel column chromatography to give5-[(S)-1-(2-nitrophenyl)-2-methyl-propyloxy]methyl-2′-deoxycytidinedC.x5 (10 mg, 96%) as a waxy solid. ¹H NMR (400 MHz, DMSO-d₆) δ 7.95 (m,1 H, Ph-H), 7.76 (m, 1 H, Ph-H), 7.64 (m, 1 H, Ph-H), 7.63 (s, 1 H,H-6), 7.56 (d, 1 H, J=8.3 Hz, Ph-H), 7.39 and 6.66 (2 br. s, 2 H, D₂Oexchangeable, NH₂), 6.13 (m, 1 H, H-1′), 5.20 (d, 1 H, J=4.1 Hz, D₂Oexchangeable, 3′-OH), 4.85 (t, 1 H, J=5.4 Hz, D₂O exchangeable, 5′-OH),4.67 (d, 1 H, J=6.0 Hz, Ph-CH), 4.17 (m, 1 H, H-3′), 4.10 (AB d, 1 H,J=12.2 Hz, 5-CH₂a), 3.99 (AB d, 1 H, J=12.2 Hz, 5-CH₂b), 3.74 (m, 1 H,H-4′), 3.49 (m, 2 H, H-5′), 2.08 (m, 1 H, H-2′a), 1.91 (m, 2 H, H-2′band CH), 0.88 (d, 3 H, J=6.7 Hz, CH₃), 0.77 (d, 3 H, J=6.9 Hz, CH₃); ¹³CNMR (100 MHz, CD₃OD): δ 166.57 (C), 158.14 (C), 151.46 (C), 142.74 (CH),137.30 (C), 134.29 (CH), 130.28 (CH), 129.91 (CH), 125.32 (CH), 104.64(C), 89.00 (CH), 87.57 (CH), 81.04 (CH), 72.13 (CH), 66.42 (CH₂), 62.87(CH₂), 42.22 (CH₂), 36.30 (CH), 19.64 (CH₃), 18.56 (CH₃).

5-[(S)-1-(2-nitrophenyl)-2-methyl-propyloxy]methyl-2′-deoxcytidine-5′-phosphate(WW3p065) POCl₃ (3 μL, 0.034 mmol) was added to a solution of compounddU.x5 (10 mg, 0.023 mmol) and proton sponge (10 mg, 0.046 mmol) intrimethylphosphate (0.3 mL) at 0° C. and stirred for two hour.Additional POCl₃ (3 μL, 0.034 mmol) was added and the mixture wasstirred for another one hour. A solution of tri-n-butylammoniumpyrophosphate (55 mg, 0.115 mmol) and tri-n-butylamine (30 μL) inanhydrous DMF (0.25 mL) was added. After five minutes of stirring,triethylammonium bicarbonate buffer (1 M, pH 7.5; 5 mL) was added. Thereaction was stirred at room temperature for one hour and thenlyophilized to dryness. The residue was dissolved in water (5 mL),filtered, and purified by anion exchange chromatography on a Q SepharoseFF column (2.5×20 cm) with a linear gradient of NH₄HCO₃ (50 mM to 500 mMin 240 minutes) at a flow rate of 4.5 mL/min. The fractions containingtriphosphate were combined and lyophilized to give5-[(S)-1-(2-nitrophenyl)-2-methyl-propyloxy]methyl-2′-deoxycytidine-5′-triphosphateWW3p065 (16 mg, 96%) as a white fluffy solid. ¹H NMR (400 MHz, D₂O): δ7.85 (dd, 1 H, J=1.2 and 8.4 Hz, Ph-H), 7.65 (m, 3 H, Ph-H and H6), 7.49(dt, 1 H, J=1.6 and 8.4 Hz, Ph-H), 6.05 (t, J=6.4 Hz, 1 H, H-1′), 4.54(AB d, 1 H, J=13.6 Hz, 5-CH₂a), 4.46 (m, 1 H, H-3′), 4.44 (AB d, 1 H,J=13.6 Hz, 5-CH₂b), 4.18 (m, 3 H, H-4′ and H-5′), 2.39 (m, 1 H, H-2′a),2.2 (m, 1 H, H-2′b), 2.01 (m, 1 H, CH), 1.06 (d, 3 H, J=6.4 Hz, CH₃),0.73 (d, 3 H, J=7.2 Hz, CH₃); ³¹P NMR (162 MHz, D₂O) for diastereomers:6-5.25 (d, J=21.0 Hz), −10.79 (d, J=19.44 Hz), −21.14 (t, J=21.0 Hz).

Example 4 Synthesis of Deoxyuridine and Deoxycytidine Analogs withα-tert-Butyl Groups Synthesis 5-[(R orS)-1-(2-nitrophenyl)-2,2-dimethyl-1-propyloxy]methyl-2′-deoxyuridine-5′-triphosphate

5-[(R orS)-1-(2-nitrophenyl)-2,2-dimethyl-1-propyloxy]methyl-2′-deoxyuridine(dU.x4): Compound dU.x0 (520 mg, 0.802 mmol) and enantio-pure (R orS)-1-(2-nitrophenyl)-2,2-dimethyl-1-propanol (580 mg, 2.77 mmol) wereheated neat at 108-115° C. for one hour under a nitrogen atmosphere. Themixture was cooled down to room temperature, dissolved in minimum amountof ethyl acetate, and purified by silica gel chromatography to yield5-[(R orS)-1-(2-nitrophenyl)-2,2-dimethyl-1-propyloxy]methyl-2′-deoxyuridine(dU.x4) (16 mg, 4%). (3′ or 5′)-O-(tert-butylsimethylsilyl)-5-[(R orS)-1-(2-nitrophenyl)-2,2-dimethyl-1-propyloxy]methyl-2′-deoxyuridine (78mg, 17%), and 3′,5′-O-bis-(tert-butylsimethylsilyl)-5-[(R orS)-1-(2-nitrophenyl)-2,2-dimethyl-1-propyl-oxy]methyl-2′-deoxyuridine(115 mg, 21%) were also obtained from the reaction. ¹H NMR (100 MHz,CD₃OD) for dU.x4: δ 7.84 (s, 1 H, H-6), 7.68 (dd, 1 H, J=1.2 and 8.1 Hz,Ph-H), 7.64 (dd, 1 H, J=1.4 and 7.9 Hz, Ph-H), 7.53 (m, 1 H, Ph-H), 7.36(m, 1 H, Ph-H), 6.13 (t, 1 H, J=7.2 Hz, H-1′), 4.84 (s, 1 H, Ph-CH),4.26 (m, 1 H, H-3′), 4.13 (AB d, 1 H, J=12.4 Hz, 5-CH₂a), 3.96 (AB d, 1H, J=12.4 Hz, 5-CH₂b), 3.78 (m, 1 H, H-4′), 3.60 (m, 2 H, H-5′), 2.12(m, 2 H, H-2′), 0.69 (s, 9H, (CH₃)₃C).

5-[(R orS)-1-(2-nitrophenyl)-2,2-dimethyl-1-propyloxy]methyl-2′-deoxyuridine-5′-triphosphate(WW3p075):POCl₃ (5 μL, 0.053 mmol) was added to a solution of compounddU.x4 (16 mg, 0.035 mmol) and proton sponge (15 mg, 0.07 mmol) intrimethylphosphate (0.3 mL) at 0° C. and stirred for two hours.Additional POCl₃ (3 μL, 0.032 mmol) was added and the mixture wasstirred for another one hour. A solution of tri-n-butylammoniumpyrophosphate (83 mg, 0.175 mmol) and tri-n-butylamine (35 μL) inanhydrous DMF (0.35 mL) was added. After five minutes of stirring,triethylammonium bicarbonate buffer (1 M, pH 7.5; 5 mL) was added. Thereaction was stirred at room temperature for one hour and thenlyophilized to dryness. The residue was dissolved in water (5 mL),filtered, and purified by anion exchange chromatography on a Q SepharoseFF column (2.5×20 cm) with a linear gradient of NH₄HCO₃ (50 mM to 500 mMin 240 minutes) at a flow rate of 4.5 mL/min. The fractions containingtriphosphate were combined and lyophilized to give 5-[(R orS)-1-(2-nitrophenyl)-2,2-dimethyl-1-propyloxy]methyl-2′-deoxyuridine-5′-triphosphateWW3p075 (14 mg, 53%) as a white fluffy solid. ¹H NMR (400 MHz, D₂O): δ7.83 (d, 1 H, J=8.8 Hz, Ph-H), 7.63 (m, 3 H, Ph-H and H-6), 7.47 (m, 1H, Ph-H), 6.08 (t, 1 H, J=6.8 Hz, H-1′), 4.46 (m, 1 H, H-3′), 4.41 (ABd, 1 H, J=8.4 Hz, 5-CH₂a), 4.32 (AB d, 1 H, J=8.8 Hz, 5-CH₂b), 4.20-4.11(m, 3 H, H-4′ and H-5′), 2.24 (m, 2 H, H-2′), 0.79 (s, 9 H, (CH₃)₃C);³¹P NMR (162 MHz, D₂O): 6-5.11 (d, J=19.4 Hz), −10.55 (d, J=19.4 Hz),−20.9 (t, J=19.4 Hz).

Synthesis 5-[(R orS)-1-(2-nitrophenyl)-2,2-dimethyl-1-propyloxy]methyl-2′-deoxycytidine-5′-triphosphate

3′,5′-O-Bis-(tert-butylsimethylsilyl)-5-[(R orS)-1-(2-nitrophenyl)-2,2-dimethyl-1-propyloxy]methyl-2′-deoxyuridine(dU.x5): Compound dU.x0 (520 mg, 0.802 mmol) and enantio-pure (R orS)-1-(2-nitrophenyl)-2,2-dimethyl-1-propanol (580 mg, 2.77 mmol) wereheated neat at 108-115° C. for one hour under a nitrogen atmosphere. Themixture was cooled down to room temperature, dissolved in minimum amountof ethyl acetate, and purified by silica gel chromatography to yield3′,5′-O-bis-(tert-butylsimethylsilyl)-5-[(R orS)-1-(2-nitrophenyl)-2,2-dimethyl-1-propyloxy]methyl-2′-deoxyuridinedU.x5 (115 mg, 21%). (3′ or 5′)-O-(tert-butylsimethylsilyl)-5-[(R orS)-1-(2-nitrophenyl)-2,2-dimethyl-1-propyloxy]methyl-2′-deoxyuridine (78mg, 17%) and 5-[(R orS)-1-(2-nitrophenyl)-2,2-dimethyl-1-propyloxy]methyl-2′-deoxyuridine (16mg, 4%) was also obtained from the reaction. ¹H NMR (400 MHz, CDCl₃) fordU.x5: δ 8.97 (s, 1 H, NH), 7.76 (d, 2 H, J=8.0 Hz, Ph-H), 7.60 (m, 2 H,Ph-H and H-6), 7.41 (s, 1 H, Ph-H), 6.29 (dd, 1 H, J=6.0 and 7.6 Hz,H-1′), 4.97 (s 1 H, Ph-CH), 4.42 (m, 1 H, H-3′), 4.28 (AB d, 1 H, J=12.0Hz, 5-CH₂a), 4.06 (AB d, 1 H, J=12.0 Hz, 5-CH₂b), 3.92 (m, 1 H, H-4′),3.76 (m, 2 H, H-5′), 2.30 (m, 1 H, H-2′a), 2.05 (m, 1 H, H-2′b), 0.95(s, 9 H, (CH₃)₃CSi), 0.90 (s, 9 H, (CH₃)₃CSi), 0.83 (s, 9 H, (CH₃)₃C),0.12 (s, 3 H, CH₃Si), 0.09 (s, 3 H, CH₃Si), 0.07 (s, 3 H, CH₃Si), 0.06(s, 3H, CH₃Si); ¹³C NMR (100 MHz, CDCl₃) for dU.x5: δ 162.52 (C), 150.82(C), 150.15 (C), 138.50 (CH), 134.19 (C), 132.01 (CH), 130.11 (CH),128.14 (CH), 123.81 (CH), 111.45 (C), 87.70 (CH), 84.98 (CH), 81.44(CH), 72.19 (CH), 64.60 (CH₂), 62.95 (CH₂), 40.85 (CH₂), 36.51 (C),25.94 ((CH₃)₃C), 25.91 ((CH₃)₃C), 25.78 (C(CH₃)₃), 18.39 (C), 18.00 (C),−4.66 (CH₃), −4.84 (CH₃), −5.35 (CH₃), −5.42 (CH₃).

3′,5′-O-Bis-(tert-butylsimethylsilyl)-5-[(R orS)-1-(2-nitrophenyl)-2,2-dimethyl-1-propyloxy]methyl-O⁴-(2,4,6-triisopropylbenzenesulfonyl)-2′-deoxyuridine(dU.x6): To a solution of dU.x5 (110 mg, 0.16 mmol), DMAP (20 mg, 0.17mmol), and triethylamine (63 μL, 0.45 mmol) in anhydrous dichloromethane(3 mL) 2,4,6-triisopropyl benzenesulfonyl chloride (61 mg, 0.20 mmol)was added. The mixture was stirred at room temperature for 36 hoursunder a nitrogen atmosphere, then concentrated in vacuo and purified bysilica gel column chromatography to give3′,5′-O-bis-(tert-butylsimethylsilyl)-5-[(R orS)-1-(2-nitrophenyl)-2,2-dimethyl-1-propyloxy]methyl-O⁴-(2,4,6-triisopropylbenzenesulfonyl)-2′-deoxyuridinedU.x6 (47 mg, 31%). ¹H NMR (500 MHz, CDCl₃): δ 8.08 (s, 1 H, H-6), 7.80(dd, 1 H, J=1.2 and 8.0 Hz, Ph-H), 7.78 (dd, 1 H, J=1.6 and 8.0 Hz,Ph-H), 7.67 (m, 1 H, Ph-H), 7.46 (m, 1 H, Ph-H), 7.20 (s, 2 H, Ph-H),6.09 (t, 1 H, J=6.4 Hz, H-1′), 4.98 (s, 1 H, Ph-CH), 4.35 (m, 1 H,H-3′), 4.25 (AB d, 1 H, J=11.6 Hz, 5-CH₂a), 4.11 (AB d, 1 H, J=11.6 Hz,5-CH₂b), 3.97 (m, 1 H, H-4′), 3.79 (dd, 1 H, J=3.6 and 11.6 Hz, H-5′a),3.74 (dd, 1 H, J=11.6 and 3.6 Hz, H-5′b), 2.90 (m, 1 H, CH), 2.50 (m, 2H, H-2′), 1.98 (m, 2 H, CH), 1.31-1.22 (m, 18 H, (CH₃)₂CH×3), 0.88 (2 s,18 H, (CH₃)₃CSi×2), 0.87 (s, 9 H, (CH₃)₃C), 0.07 (s, 6 H, (CH₃)₂Si),0.06 (s, 6 H, (CH₃)₂Si); ¹³C NMR (100 MHz, CDCl₃): δ 166.03 (C), 154.39(C), 153.52 (C), 151.09 (C), 150.94 (C), 144.77 (CH), 133.60 (C), 132.43(CH), 131.07 (C), 130.12 (CH), 128.24 (CH), 123.05 (CH), 123.81 (CH),104.64 (C), 88.39 (CH), 87.42 (CH), 82.45 (CH), 71.93 (CH), 64.41 (CH₂),62.78 (CH₂), 42.09 (CH₂), 36.54 (C), 34.26 (CH), 29.60 (CH), 25.92((CH₃)₃C), 25.82 ((CH₃)₃C), 25.74 ((CH₃)₃C), 24.62 (CH₃), 24.34 (CH₃),23.48 (CH₃), 18.40 (C), 17.99 (C), −4.62 (CH₃), −4.92 (CH₃), −5.37(CH₃), −5.34 (CH₃).

3′,5′-O-Bis-(tert-butyldimethylsilyl)-5-[(R orS)-1-(2-nitrophenyl)-2,2-dimethyl-1-propyloxy]methyl-2′-deoxcytidine(dC.x6): To a solution of compound dU.x6 (47 mg, 0.050 mmol) inanhydrous 1,4-dioxane (2 mL) a solution of ammonia (2 mL, 0.5 M indioxane) was added. The mixture was transferred into a sealable tube andwas heated at 92° C. for 10 hours. The mixture was cooled down to roomtemperature, concentrated in vacuo and purified by silica gel columnchromatography to yield 3′,5′-O-bis-(tert-butyldimethylsilyl)-5-[(R orS)-1-(2-nitrophenyl)-2,2-dimethyl-1-propyloxy]methyl-2′-deoxcytidinedC.x6 (31 mg, 91%). ¹H NMR (400 MHz, CDCl₃) δ 7.67 (m, 3 H, Ph-H), 7.53(s, 1 H, H-6), 7.45 (m, 1 H, Ph-H), 6.30 (t, 1 H, J=6.6 Hz, H-1′), 5.72(br s, 2 H, NH₂), 4.88 (s, 1 H, Ph-CH), 4.32 (m, 1 H, H-3′), 4.28 (AB d,1 H, J=12.8 Hz, 5-CH₂a), 4.08 (AB d, 1 H, J=12.8 Hz, 5-CH₂b), 3.87 (m, 1H, H-4′), 3.74 (dd, 1 H, J=3.6 and 14.8 Hz, H-5′a), 3.66 (dd, 1 H, J=3.6and 11.3 Hz, H-5′b), 2.41 (m, 1 H, H-2′a), 2.03 (m, 1 H, H-2′b), 0.90(s, 9 H, (CH₃)₃CSi), 0.87 (s, 9 H, (CH₃)₃CSi), 0.83 (s, 9 H, (CH₃)₃C),0.09 (2 s, 6 H, (CH₃)₂Si), 0.06 (2 s, 6 H, (CH₃)₂Si); ¹³C NMR (100 MHz,CDCl₃): δ 165.16 (C), 155.52 (C), 151.31 (C), 140.63 (CH), 133.31 (C),132.04 (CH), 129.53 (CH), 128.56 (CH), 123.76 (CH), 101.89 (C), 87.39(CH), 85.72 (CH), 81.24 (CH), 71.59 (CH), 66.55 (CH₂), 62.68 (CH₂),41.65 (CH₂), 36.20 (C), 25.92 ((CH₃)₃C), 25.82 ((CH₃)₃C), 25.75((CH₃)₃C), 18.23 (C), 17.97 (C), −4.64 (CH₃), −4.94 (CH₃), −5.47 (CH₃),−5.53 (CH₃).

5-[(R orS)-1-(2-nitrophenyl)-2,2-dimethyl-1-propyloxy]methyl-2′-deoxcytidine(dC.x7): A solution of tetra-n-butylammonium fluoride trihydrate (28 mg,0.09 mmol) in THF (1 mL) was added to a solution of compound dC.x6 (20mg, 0.03 mmol) in THF (2 mL). The mixture was stirred at roomtemperature for 30 minutes, then concentrated in vacuo and purified bysilica gel column chromatography to yield 5-[(R orS)-1-(2-nitrophenyl)-2,2-dimethyl-1-propyloxy]methyl-2′-deoxcytidinedC.x7 (11 mg, 82%). ¹H NMR (400 MHz, CD₃OD) δ 7.87 (s, 1 H, H-6), 7.82(dd, 1 H, J=1.2 and 8.4 Hz, Ph-H), 7.76 (dd, 1 H, J=1.6 and 8.0 Hz,Ph-H), 7.68 (m, 1 H, Ph-H), 7.51 (m, 1 H, Ph-H), 6.23 (t, 1 H, J=6.6 Hz,H-1′), 4.94 (s, 1 H, Ph-CH), 4.44 (AB d, 1 H, J=13.2 Hz, 5-CH₂a), 4.34(m, 1 H, H-3′), 4.11 (AB d, 1 H, J=13.2 Hz, 5-CH₂b), 3.88 (m, 1 H,H-4′), 3.71 (dd, 1 H, J=3.2 and 12.0 Hz, H-5′a), 3.63 (dd, 1 H, J=4.0and 12.0 Hz, H-5′b), 2.35 (m, 1 H, H-2′a), 2.14 (m, 1 H, H-2′b), 0.80(s, 9 H, (CH₃)₃C); ¹³C NMR (100 MHz, CD₃OD): δ 166.65 (C), 158.22 (C),152.66 (C), 143.25 (CH), 134.69 (C), 133.42 (CH), 131.19 (CH), 129.96(CH), 125.30 (CH), 104.49 (C), 88.94 (CH), 87.46 (CH), 81.44 (CH), 72.20(CH), 66.33 (CH₂), 62.88 (CH₂), 42.11 (CH₂), 37.34 (C), 26.44 ((CH₃)₃C).

5-[(R orS)-1-(2-nitrophenyl)-2,2-dimethyl-1-propyloxy]methyl-2′-deoxcytidine-5′triphosphate(WW3p085): POCl₃ (3.5 μL, 0.038 mmol) was added to a solution ofcompound dU.x7 (11 mg, 0.025 mmol) and proton sponge (10.5 mg, 0.049mmol) in trimethylphosphate (0.3 mL) at 0° C. and stirred for two hour.Additional POCl₃ (3.5 μL, 0.038 mmol) was added and the mixture wasstirred for another one hour. A solution of tri-n-butylammoniumpyrophosphate (59 mg, 0.125 mmol) and tri-n-butylamine (30 μL) inanhydrous DMF (0.25 mL) was added. After five minutes of stirring,triethylammonium bicarbonate buffer (1 M, pH 7.5; 5 mL) was added. Thereaction was stirred at room temperature for one hour and thenlyophilized to dryness. The residue was dissolved in water (5 mL),filtered, and purified by anion exchange chromatography on a Q SepharoseFF column (2.5×20 cm) with a linear gradient of NH₄HCO₃ (50 mM to 500 mMin 240 minutes) at a flow rate of 4.5 mL/min. The fractions containingtriphosphate were combined and lyophilized to give 5-[(R orS)-1-(2-nitrophenyl)-2,2-dimethyl-1-propyloxy]methyl-2′-deoxycytidine-5′-triphosphateWW3p085 (12 mg, 65%) as a white fluffy solid. ¹H NMR (400 MHz, CD₃OD) δ7.81 (d, 1 H, J=8.0 Hz, Ph-H), 7.65 (m, 3 H, Ph-H and H-6), 7.45 (t, 1H, J=8.0 Hz, Ph-H), 6.03 (t, 1 H, J=6.4 Hz, H-1′), 4.52 (AB d, 1 H,J=13.6 Hz, 5-CH₂a), 4.46 (d, 1 H, J=13.6 Hz, 5-CH₂b), 4.41 (m, 1 H,H-3′), 4.12 (m, 3 H, H-4′ and H-5′), 2.35 (m, 1 H, H-2′a), 2.17 (m, 1 H,H-2′b), 0.82 (s, 9 H, (CH₃)₃C); ³¹P NMR (162 MHz, D₂O) fordiastereomers: 6-5.15 (d, J=21.0 Hz), −10.64 (d, J=19.44 Hz), −21.0 (t,J=21.0 Hz).

Example 5 Synthesis of Dye-Attached Deoxyuridine and DeoxycytidineAnalogs with α-Isopropyl Groups Synthesis of 6-JOE labeled5-{(R)-1-[4-(3-amino-1-propynyl)-2-nitrophenyl]-2-methyl-propyloxy}methyl-2′-deoxyuridine-5′-triphosphate

5-[(R)-1-(4-Iodo-2-nitrophenyl)-2,2-dimethylpropyloxy]methyl-2′-deoxyuridine(dU.y1): Compound dU.x0 (775 mg, 1.19 mmol) and enantio-pure(R)-1-(4-iodo-2-nitrophenyl)-2-methyl-1-propanol (1.22 g, 3.80 mmol)were heated neat at 108-112° C. for 45 minutes under a nitrogenatmosphere. The mixture was cooled down to room temperature, dissolvedin minimum amount of ethyl acetate, and purified by silica gelchromatography to yield5-[(R)-1-(4-iodo-2-nitrophenyl)-2-methyl-propyloxy]methyl-2′-deoxyuridine(dU.y1) (60 mg, 9%). (3′ or5′)-O-(tert-butylsimethylsilyl)-5-[(R)-1-(4-iodo-2-nitrophenyl)-2-methyl-propyloxy]methyl-2′-deoxyuridine(90 mg, 11%) and3′,5′-O-bis-(tert-butylsimethylsilyl)-5-[(R)-1-(4-iodo-2-nitrophenyl)-2-methyl-propyl-oxy]methyl-2′-deoxyuridine(194 mg, 21%) were also obtained from the reaction. ¹H NMR (400 MHz,CD₃CD) for dU.y1: δ 8.23 (d, 1 H, J=1.7 Hz, Ph-H), 8.03 (dd, J=8.4 and1.7 Hz, 1 H, Ph-H), 8.00 (s, 1 H, H-6), 7.50 (d, 1 H, J=8.4 Hz, Ph-H),6.26 (t, 1 H, J=6.8 Hz, H-1′), 4.69 (d, 1 H, J=5.8 Hz, PhCH), 4.40 (m, 1H, H-3′), 4.13 (AB d, J=11.8 Hz, 1 H, 5-CH₂a), 4.08 (AB d, J=11.8 Hz, 1H, 5-CH₂b), 3.93 (m, 1 H, H-4′), 3.79 (dd, J=12.0 and 3.3 Hz, 1 H,H-5′a), 3.73 (dd, J=12.0 and 3.6 Hz, 1 H, H-5′b), 2.26 (m, 1 H, H-2′a),2.19 (m, 1 H, H-2′b), 1.93 (m, 1 H, CH(CH₃)₃), 0.93 (d, J=6.4 Hz, 3H,CH₃), 0.87 (d, J=6.8 Hz, 3 H, CH₃).

5-{(R)-1-[4-(3-Trifluoroacetamido-1-propynyl)-2-nitrophenyl]-2-methyl-propyloxy}methyl-2′-deoxyuridine(dU.y2): A solution of compound dU.y1 (60 mg, 0.11 mmol),N-propargyltrifluoroacetylamide (48 mg, 0.32 mmol),tetrakis(triphenyl-phosphine)-palladium(0) (12 mg, 0.01 mmol), CuI (4mg, 0.02 mmol), and Et₃N (30 μL, 0.21 mmol) in anhydrous DMF (5 mL) wasstirred at room temperature for 4.5 hours. Methanol (4 mL) and methylenechloride (4 mL) were added, followed by sodium bicarbonate (49 mg, 0.58mmol). The mixture was stirred for another hour, then concentrated invacuo and purified by silica gel column chromatography to yield5-{(R)-1-[4-(3-trifluoroacetamido-1-propynyl)-2-nitrophenyl]-2-methyl-propyloxy}methyl-2′-deoxyuridinedU.y2 (54 mg, 86%). ¹H NMR (400 MHz, DMSO-d₆): δ 11.33 (s, 1 H, D₂Oexchangeable, 3-NH), 10.12 (br t, J=4.8 Hz, 1H, D₂O exchangeable,CH₂NHTFA), 7.98 (d, 1 H, J=1.5 Hz, Ph-H), 7.85 (s, 1 H, H-6), 7.74 (dd,J=8.2 and 1.5 Hz, 1 H, Ph-H), 7.64 (d, 1 H, J=8.2 Hz, Ph-H), 6.13 (t, 1H, J=6.8 Hz, H-1′), 5.24 (d, J=4.2 Hz, 1 H, D₂O exchangeable, 3′-OH),4.96 (t, J=5.2 Hz, 1H, D₂O exchangeable, 5′-OH), 4.61 (d, 1 H, J=5.5 Hz,PhCH), 4.30 (d, 2 H, J=4.8 Hz, CH ₂NHTFA), 4.22 (m, 1 H, H-3′), 3.97 (s,2 H, 5-CH₂a and 5-CH₂b), 3.76 (m, 1 H, H-4′), 3.56 (m, 2 H, H-5′a andH-5′b), 2.06 (m, 2 H, H-2′a and H-2′b), 1.88 (m, 1 H, CH(CH₃)₃), 0.83(d, J=6.7 Hz, 3 H, CH₃), 0.80 (d, J=6.8 Hz, 3 H, CH₃); ¹³C NMR (100 MHz,CD₃OD): δ 163.59 (C), 157.22 (C), 150.67 (C), 149.23 (C), 139.78 (CH),137.05 (C), 135.21 (CH), 129.48 (CH), 126.63 (CH), 122.76 (C), 110.89(C), 87.60 (CH), 85.70 (C), 85.14 (CH), 87.96 (CH), 80.27 (C), 70.86(CH), 64.34 (CH₂), 61.44 (CH₂), 39.96 (CH₂), 34.62 (CH), 29.07 (CH₂),18.30 (CH₃), 16.44 (CH₃).

5-{(R)-1-[4-(3-Amino-1-propynyl)-2-nitrophenyl]-2-methyl-propyloxy}methyl-2′-deoxyuridine-5′-triphosphate(dU.y3): POCl₃ (6 μL, 0.06 mmol) was added to a solution of compounddU.y2 (18 mg, 0.03 mmol) and proton sponge (13 mg, 0.06 mmol) intrimethylphosphate (0.3 mL) at 0° C. and stirred for two hours. Asolution of bis-tri-n-butylammonium pyrophosphate (73 mg, 0.15 mmol) andtri-n-butylamine (30 μL) in anhydrous DMF (0.3 mL) was added. After fiveminutes of stirring, triethylammonium bicarbonate buffer (1 M, pH 7.5; 5mL) was added. The reaction was stirred for one hour at room temperatureand then lyophilized to dryness. The residue was dissolved in water (5mL), filtered, and part of the solution was purified with reverse-phaseHPLC using a Perkin Elmer OD-300 C₁₈ column (4.6×250 mm) to yield5-{(R)-1-[4-(3-trifluoroacetamido-1-propynyl)-2-nitrophenyl]-2-methyl-propyloxy}methyl-2′-deoxy-uridine-5′-trihosphate.Mobile phase: A, 100 mM triethylammonium acetate (TEAA) in water (pH7.0); B, 100 mM TEAA in water/CH₃CN (30:70). The purified triphosphatewas then treated with concentrated ammonium hydroxide (27%) at roomtemperature for two hours to yield5-{(R)-1-[4-(3-amino-1-propynyl)-2-nitrophenyl]-2-methyl-propyloxy}methyl-2′-deoxyuridine-5′-triphosphatedU.y3. ¹H NMR (400 MHz, D₂O): δ 8.01 (s, 1 H, Ph-H), 7.76 (d, 1 H, J=6.9Hz, Ph-H), 7.62 (m, 2 H, H-6 and Ph-H), 6.17 (t, 1 H, J=6.4 Hz, H-1′),4.55 (m, 1 H, H-3′), 4.39 and 4.29 (2 d, 2 H, J=6.4 Hz, CH₂), 4.17 (m, 3H, H-4′ and H-5′), 3.74 (s, 2 H, CH₂), 2.28 (m, 2 H, H-2′), 2.00 (m, 1H, CH), 0.79 (m, 3 H, CH₃); ³¹P NMR (162 MHz, D₂O): 6-5.40 (d, J=19.4Hz), −10.75 (d, J=19.4 Hz), −21.23 (t, J=19.4 Hz).

6-JOE labeled5-{(R)-1-[4-(3-amino-1-propynyl)-2-nitrophenyl]-2-methyl-propyloxy}methyl-2′-deoxyuridine-5′-triphosphate(WW3p024): A solution of 6-JOE-SE (0.625 mg, 1 μmol) in anhydrous DMSO(25 μL) was added to a solution of triphosphate dU.y3 (0.31 μmol) inNa₂CO₃/NaHCO₃ buffer (0.1 M, pH 9.2; 180 μL) and incubated at roomtemperature for one hour. The reaction was purified by reverse-phaseHPLC using a Perkin Elmer OD-300 C₁₈ column (4.6×250 mm) to yield the6-JOE labeled triphosphate WW3p024. Mobile phase: A, 100 mM TEAA inwater (pH 7.0); B, 100 mM TEAA in water/CH₃CN (30:70). WW3p024 wascharacterized by its incorporation by DNA polymerase and photodeprotection.

Synthesis of Cy5 labeled5-{(R)-1-[4-(3-amino-1-propynyl)-2-nitrophenyl]-2-methyl-propyloxy}methyl-2′-deoxycytidine-5′-triphosphate

3′,5′-O-Bis-(tert-butyldimethylsilyl)-5-[(R)-1-(4-iodo-2-nitrophenyl)-2-methyl-propyloxy]methyl-2′-deoxyuridine(dU.y4): Compound dU.x0 (775 mg, 1.19 mmol) and enantio-pure(R)-1-(4-iodo-2-nitrophenyl)-2-methyl-1-propanol (1.22 g, 3.80 mmol)were heated neat at 108-112° C. for 45 minutes under a nitrogenatmosphere. The mixture was cooled down to room temperature, dissolvedin minimum amount of ethyl acetate, and purified by silica gelchromatography to yield3′,5′-O-bis-(tert-butyldimethylsilyl)-5-[(R)-1-(4-iodo-2-nitrophenyl)-2-methyl-propyloxy]methyl-2′-deoxyuridinedU.y4 (194 mg, 21%). (3′ or5′)-O-(tert-butyldimethylsilyl)-5-[(R)-1-(4-iodo-2-nitrophenyl)-2-methyl-propyloxy]methyl-2′-deoxyuridine(90 mg, 11%) and5-[(R)-1-(4-iodo-2-nitrophenyl)-2-methyl-propyloxy]methyl-2′-deoxyuridine(60 mg, 9%) were also obtained from the reaction. ¹H NMR (400 MHz,CDCl₃) for dU.y4: δ 8.43 (s, 1 H, 3-NH), 8.18 (d, 1 H, J=1.6 Hz, Ph-H),7.96 (dd, J=8.3 and 1.6 Hz, 1 H, Ph-H), 7.65 (s, 1 H, H-6), 7.47 (d, 1H, J=8.3 Hz, Ph-H), 6.29 (dd, 1 H, J=7.8 and 5.8 Hz, H-1′), 4.74 (d, 1H, J=5.8 Hz, PhCH), 4.39 (m, 1 H, H-3′), 4.14 (AB d, J=11.6 Hz, 1 H,5-CH₂a), 3.98 (AB d, J=11.6 Hz, 1 H, 5-CH₂b), 3.97 (m, 1 H, H-4′), 3.78(m, 2 H, H-5′a and H-5′b), 2.31 (m, 1 H, H-2′a), 1.99 (m, 1 H, H-2′b),1.91 (m, 1 H, CH(CH₃)₃), 0.92 (d, J=6.7 Hz, 3 H, CH₃), 0.90 (s, 9 H,(CH₃)₃CSi), 0.98 (s, 9 H, (CH₃)₃CSi), 0.86 (d, J=6.9 Hz, 3H, CH₃), 0.09(s, 3 H, CH₃Si), 0.08 (s, 3 H, CH₃Si), 0.07 (s, 3 H, CH₃Si), 0.04 (s, 3H, CH₃Si); ¹³C NMR (100 MHz, CDCl₃) for dU.y4: δ 162.20 (C), 149.83 (C),149.69 (C), 141.89 (CH), 138.68 (CH), 136.56 (C), 132.63 (CH), 130.94(CH), 111.19 (C), 91.84 (C), 88.00 (CH), 85.41 (CH), 80.99 (CH), 72.40(CH), 64.46 (CH₂), 63.18 (CH₂), 41.22 (CH₂), 34.74 (CH), 25.93(C(CH₃)₃), 25.75 (C(CH₃)₃), 19.29 (CH₃), 18.40 (C), 18.00 (C), 17.49(CH₃), −4.64 (CH₃), −4.81 (CH₃), −5.37 (2 CH₃).

3′,5-O-Bis-(tert-butyldimethylsilyl)-5-[(R)-1-(4-iodo-2-nitrophenyl)-2-methyl-propyloxy]methyl-2′-deoxcytidine(dC.y5): To a solution of compound dU.y4 (0.256 g, 0.32 mmol), DMAP (23mg, 0.21 mmol) and triethylamine (1.138 mL, 8.10 mmol) in anhydrousdichloromethane (14 mL), 2,4,6-triisopropyl benzenesulfonyl chloride(1.57 g, 5.19 mmol) was added. The mixture was stirred at roomtemperature for 16 hours under a nitrogen atmosphere, and thenconcentrated in vacuo. Ammonia (0.5 M in dioxane, 24 mL, 12.0 mmol) wasadded and the mixture was transferred into a sealed tube, and heated at92-94° C. for four hours. The mixture was concentrated under reducedpressure and purified by silica gel column chromatography to yield3′,5′-O-bis-(tert-butyldimethylsilyl)-5-[(R)-1-(4-iodo-2-nitrophenyl)-2-methyl-propyloxy]methyl-2′-deoxcytidinedC.y5 (192 mg, 76%). ¹H NMR (400 MHz, CDCl₃) δ 8.13 (d, 1 H, J=1.6 Hz,Ph-H), 7.95 (dd, J=8.3 and 1.6 Hz, 1 H, Ph-H), 7.59 (s, 1 H, H-6), 7.38(d, 1 H, J=8.3 Hz, Ph-H), 6.28 (t, 1 H, J=6.5 Hz, H-1′), 6.02 (br, 2 H,4-NH₂), 4.63 (d, 1 H, J=6.6 Hz, PhCH), 4.32 (m, 1 H, H-3′), 4.19 (AB d,J=12.5 Hz, 1H, 5-CH₂a), 4.04 (AB d, J=12.5 Hz, 1 H, 5-CH₂b), 3.93 (m, 1H, H-4′), 3.81 (AB dd, J=11.3 and 3.0 Hz, 1 H, H-5′a), 3.72 (AB dd,J=11.3 and 2.8 Hz, 1 H, H-5′b), 2.43 (m, 1 H, H-2′a), 1.92 (m, 2 H,H-2′b and CH(CH₃)₃), 0.96 (d, J=6.6 Hz, 3H, CH₃), 0.82 (d, J=6.9 Hz, 3H,CH₃), 0.89 (s, 9 H, (CH₃)₃CSi), 0.80 (s, 9 H, (CH₃)₃CSi), 0.06 (s, 3 H,CH₃Si), 0.05 (s, 3 H, CH₃Si), −0.02 (s, 3 H, CH₃Si), −0.04 (s, 3 H,CH₃Si); ¹³C NMR (100 MHz, CDCl₃): δ 165.02 (C), 152.93 (C), 150.09 (C),141.92 (CH), 140.37 (CH), 135.53 (C), 132.55 (CH), 130.35 (CH), 101.50(C), 92.35 (C), 87.76 (CH), 86.26 (CH), 80.33 (CH), 71.98 (CH), 66.92(CH₂), 62.84 (CH₂), 42.14 (CH₂), 34.75 (CH), 25.89 (C(CH₃)₃), 25.77(C(CH₃)₃), 19.01 (CH₃), 18.30 (C), 18.15 (CH₃), 18.00 (C), −4.57 (CH₃),−4.87 (CH₃), −5.43 (CH₃), −5.49 (CH₃).

5-[(R)-1-(4-Iodo-2-nitrophenyl)-2-methyl-propyloxy]methyl-2′-deoxcytidinedC.y6: A solution of tetra-n-butylammonium fluoride trihydrate (112 mg,0.36 mmol) in THF (2 mL) was added to a solution of compound dC.y5 (192mg, 0.24 mmol) in THF (8 mL). The mixture was stirred at roomtemperature for 45 minutes, then concentrated in vacuo and purified bysilica gel column chromatography to yield5-[(R)-1-(4-iodo-2-nitrophenyl)-2-methyl-propyloxy]methyl-2′-deoxcytidinedC.y6 (110 mg, 80%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.26 (d, 1 H, J=1.7 Hz,Ph-H), 8.07 (dd, J=8.3 and 1.7 Hz, 1 H, Ph-H), 7.70 (s, 1 H, H-6), 7.38(d, 1 H, J=8.3 Hz, Ph-H), 7.32 and 6.59 (2 br. s, 2 H, D₂O exchangeable,4-NH₂), 6.11 (dd, 1 H, J=7.2 and 6.1 Hz, H-1′), 5.19 (d, J=4.2 Hz, 1 H,D₂O exchangeable, 3′-OH), 4.88 (t, J=5.4 Hz, 1 H, D₂O exchangeable,5′-OH), 4.58 (d, 1 H, J=6.0 Hz, PhCH), 4.19 (m, 1 H, H-3′), 4.07 (AB d,J=12.2 Hz, 1 H, 5-CH₂a), 4.02 (AB d, J=12.2 Hz, 1 H, 5-CH₂b), 3.75 (m, 1H, H-4′), 3.51 (m, 2 H, H-5′a and H-5′b), 2.09 (m, 1 H, H-2′a), 1.90 (m,2 H, H-2′b and CH(CH₃)₃), 0.87 (d, J=6.7 Hz, 3 H, CH₃), 0.78 (d, J=6.8Hz, 3 H, CH₃); ¹³C NMR (100 MHz, CD₃OD): δ 166.48 (C), 158.09 (C),151.27 (C), 143.22 (CH), 142.77 (CH), 137.32 (C), 133.80 (CH), 132.20(CH), 104.72 (C), 93.10 (C), 89.08 (CH), 87.73 (CH), 81.82 (CH), 72.05(CH), 67.22 (CH₂), 62.82 (CH₂), 42.21 (CH₂), 36.20 (CH), 19.62 (CH₃),18.41 (CH₃).

5-{(R)-1-[4-(3-Trifluoroacetamido-1-propynyl)-2-nitrophenyl]-2-methyl-propyloxy}methyl-2′-deoxcytidine(dC.y7): A solution of compound dC.y6 (110 mg, 0.20 mmol),N-propargyltrifluoroacetylamide (88 mg, 0.59 mmol),tetra-kis(triphenylphosphine)-palladium(0) (23 mg, 0.02 mmol), CuI (7mg, 0.04 mmol), and Et₃N (0.11 mL, 0.78 mmol) in anhydrous DMF (4 mL)was stirred at room temperature for 4.5 hours. Methanol (4 mL) andmethylene chloride (4 mL) were added, followed by sodium bicarbonate (90mg, 1.07 mmol). The mixture stirred for another one hour, thenconcentrated in vacuo and purified by silica gel column chromatographyto yield5-{(R)-1-[4-(3-trifluoroacetamido-1-propynyl)-2-nitrophenyl]-2-methyl-propyloxy}methyl-2′-deoxcytidinedC.y7 (112 mg, 99%). ¹H NMR (400 MHz, DMSO-d₆): δ 10.12 (br t, 1 H, D₂Oexchangeable, CH₂NHTFA), 7.98 (d, 1 H, J=1.6 Hz, Ph-H), 7.76 (dd, J=8.2and 1.7 Hz, 1 H, Ph-H), 7.73 (s, 1 H, H-6), 7.62 (d, 1 H, J=8.2 Hz,Ph-H), 7.33 and 6.60 (2 br. s, 2 H, D₂O exchangeable, 4-NH₂), 6.11 (t, 1H, J=6.8 Hz, H-1′), 5.19 (d, J=4.2 Hz, 1 H, D₂O exchangeable, 3′-OH),4.89 (t, J=5.4 Hz, 1 H, D₂O exchangeable, 5′-OH), 4.64 (d, 1 H, J=5.9Hz, PhCH), 4.31 (d, 2 H, J=5.2 Hz, CH ₂NHTFA), 4.19 (m, 1 H, H-3′), 4.09(AB d, J=12.1 Hz, 1 H, 5-CH₂a), 4.03 (AB d, J=12.1 Hz, 1 H, 5-CH₂b),3.75 (m, 1 H, H-4′), 3.52 (m, 2 H, H-5′a and H-5′b), 2.08 (m, 1 H,H-2′a), 1.91 (m, 2 H, H-2′ b and CH(CH₃)₃), 0.87 (d, J=6.7 Hz, 3 H,CH₃), 0.79 (d, J=6.8 Hz, 3 H, CH₃): ¹³C NMR (100 MHz, CD₃OD): δ 164.83(C), 157.51 (C), 157.14 (C), 156.45 (C), 149.25 (C), 141.23 (CH), 136.46(C), 135.39 (CH), 129.39 (C), 126.64 (CH), 122.98 (C), 117.43 (C),114.58 (C), 87.54 (CH), 86.22 (CH), 86.02 (C), 80.34 (C), 80.27 (CH),70.53 (CH), 65.75 (CH₂), 61.28 (CH₂), 40.60 (CH₂), 34.73 (CH), 29.16(CH₂), 18.15 (CH₃), 16.96 (CH₃). HRMS: for C₂₅H₂₉F₃N₅O₈ [MH⁺]: clcd584.1968 found 584.1926

5-{(R)-1-[4-(3-Amino-1-propynyl)-2-nitrophenyl]-2-methyl-propyloxy}methyl-2′-deoxcytidine-5′-triphosphate(dC.y8): POCl₃ (6 μL, 0.06 mmol) was added to a solution of compounddC.y7 (19 mg, 0.03 mmol) and proton sponge (14 mg, 0.06 mmol) intrimethylphosphate (0.3 mL) at 0° C. and stirred for two hours. Asolution of bis-tri-n-butylammonium pyrophosphate (76 mg, 0.16 mmol) andtri-n-butylamine (32 μL) in anhydrous DMF (0.32 mL) was added. Afterfive minutes of stirring, triethylammonium bicarbonate buffer (1 M, pH7.5; 5 mL) was added. The reaction was stirred for one hour at roomtemperature and then concentrated in vacuo at 25° C. The residue wasdissolved in water (2 mL), filtered, and purified with reverse-phaseHPLC using a Perkin Elmer OD-300 C₁₈ column (4.6×250 mm) to yield5-{(R)-1-[4-(3-trifluoroacetamido-1-propynyl)-2-nitrophenyl]-2-methyl-propyloxy}methyl-2′-deoxycytidine-5′-trihosphate.Mobile phase: A, 100 mM triethylammonium acetate (TEAA) in water (pH7.0); B, 100 mM TEAA in water/CH₃CN (30:70). The purified triphosphatewas then treated with concentrated ammonium hydroxide (27%) at roomtemperature for two hours to yield5-{(R)-1-[4-(3-amino-1-propynyl)-2-nitrophenyl]-2-methyl-propyloxy}methyl-2′-deoxycytidine-5′-triphosphatedC.y8.

Cy5 labeled5-{(R)-1-[4-(3-amino-1-propynyl)-2-nitrophenyl]-2-methyl-propyl-oxy}methyl-2′-deoxycytidine-5′-triphosphate(WW3p117): A solution of Cy5 mono NHS (1 mg, 1.26 μmol) in anhydrousDMSO (40 μL) was added to a solution of triphosphate dC.y8 (0.31 μmol)in Na₂CO₃/NaHCO₃ buffer (0.1 M, pH 9.2; 100 μL) and left at roomtemperature for one hour. The reaction was purified by reverse-phaseHPLC using a Perkin Elmer OD-300 C₁₈ column (4.6×250 mm) to yield theCy5 labeled triphosphate WW3p117. Mobile phase: A, 100 mM TEAA in water(pH 7.0); B, 100 mM TEAA in water/CH₃CN (30:70).

Example 6 Synthesis of Dye-Attached Deoxyuridine and DeoxycytidineAnalogs with α-tert-Butyl Groups Synthesis of 6-TAMRA labeled 5-{(R orS)-1-[4-(3-amino-1-propynyl)-2-nitrophenyl]-2,2-dimethyl-propoxy}methyl-2′-deoxycytidine-5′-triphosphate

3′,5′-O-Bis-(tert-butyldimethylsilyl)-5-[(R orS)-1-(4-iodo-2-nitrophenyl)-2,2-dimethyl-propyloxy]methyl-2′-deoxyuridine(dU.z1): Compound dU.x0 (688 mg, 1.06 mmol) and enantio-pure (R orS)-1-(4-iodo-2-nitrophenyl)-2,2-dimethyl-1-propanol (889 mg, 2.65 mmol)were heated neat at 108-115° C. for one hour under a nitrogenatmosphere. The mixture was cooled down to room temperature, dissolvedin minimum amount of ethyl acetate, and purified by silica gelchromatography to yield 3′,5′-O-bis-(tert-butyldimethylsilyl)-5-[(R orS)-1-(4-iodo-2-nitrophenyl)-2,2-dimethyl-propyloxy]-methyl-2′-deoxyuridinedU.z1 (236 mg, 28%). (3′ or 5′)-O-(tert-butyldimethylsilyl)-5-[(R orS)-1-(4-Iodo-2-nitrophenyl)-2,2-dimethyl-propyloxy]methyl-2′-deoxyuridine(20 mg, 3%) and 5-[(R orS)-1-(4-iodo-2-nitrophenyl)-2,2-dimethyl-propyloxy]methyl-2′-deoxyuridine(49 mg, 8%) were also obtained from the reaction. ¹H NMR (400 MHz,CDCl₃) for dU.z1: δ 8.86 (s, 1 H, 3-NH), 8.08 (d, 1 H, J=1.8 Hz, Ph-H),7.94 (dd, J=8.4 and 1.7 Hz, 1 H, Ph-H), 7.68 (s, 1 H, H-6), 7.47 (d, 1H, J=8.4 Hz, Ph-H), 6.29 (dd, 1 H, J=7.8 and 5.8 Hz, H-1′), 4.91 (s, 1H, PhCH), 4.39 (m, 1 H, H-3′), 4.23 (AB d, J=11.8 Hz, 1 H, 5-CH₂a), 4.01(AB d, J=11.8 Hz, 1 H, 5-CH₂b), 3.98 (m, 1 H, H-4′), 3.78 (m, 2 H, H-5′aand H-5′b), 2.31 (m, 1 H, H-2′a), 1.99 (m, 1 H, H-2′b), 1.91 (m, 1 H,CH(CH₃)₃), 0.90 (s, 9 H, (CH₃)₃CSi), 0.89 (s, 9 H, (CH₃)₃CSi), 0.82 (s,9 H, (CH₃)₃CC), 0.09 (2 s, 6 H, (CH₃)₂Si), 0.08 (s, 3 H, CH₃Si), 0.06(s, 3 H, CH₃Si); ¹³C NMR (100 MHz, CDCl₃) for dU.z1: δ 162.41 (C),150.98 (C), 150.00 (C), 141.60 (CH), 138.58 (CH), 133.83 (C), 132.26(CH), 131.89 (CH), 111.16 (C), 92.05 (C), 88.03 (CH), 85.49 (CH), 81.62(CH), 72.46 (CH), 64.64 (CH₂), 63.22 (CH₂), 41.22 (CH₂), 36.55 (C),25.92 (C(CH₃)₃), 25.75 (C(CH₃)₃), 25.70 (C(CH₃)₃), 18.38 (C), 18.00 (C),−4.64 (CH₃Si), −4.81 (CH₃Si), −5.39 (2 (CH₃)₂Si).

3′,5′-O-Bis-(tert-butyldimethylsilyl)-5-[(R orS)-1-(4-iodo-2-nitrophenyl)-2,2-di-methyl-propyloxy]methyl-O⁴-(2,4,6-triisopropylbenzenesulfonyl)-2′-deoxyuridine(dU.z2): 2,4,6-Triisopropyl benzenesulfonyl chloride (1.57 g, 5.19 mmol)was added to a solution of compound dU.z1 (0.236 g, 0.29 mmol), DMAP (38mg, 0.32 mmol) and triethylamine (0.465 mL, 3.31 mmol) in anhydrousdichloromethane (10 mL). The mixture was stirred at room temperature for24 hours under a nitrogen atmosphere, then concentrated in vacuo andpurified by silica gel column chromatography to give3′,5′-β-bis-(tert-butyldimethylsilyl)-5-[(R orS)-1-(4-iodo-2-nitrophenyl)-2,2-dimethyl-propyl-oxy]methyl-O⁴-(2,4,6-triisopropylbenzenesulfonyl)-2′-deoxyuridinedU.z2 (169 mg, 54%). ¹H NMR (500 MHz, CDCl₃): δ 8.18 (s, 1 H, H-6), 8.10(d, J=1.8 Hz, 1 H, Ph-H), 7.98 (dd, J=8.3 and 1.8 Hz, 1 H, Ph-H), 7.63(d, J=8.3 Hz, 1 H, Ph-H), 7.20 (s, 2 H, OSO₂Ph-H), 6.10 (t, J=6.3 Hz, 1H, H-1′), 4.91 (s, 1 H, PhCH(t-Bu)O), 4.30 (m, 1 H, H-3′), 4.23 (m 3 H,5-CH₂a and OSO₂Ph(o-CH(CH₃)₂)₂), 4.01 (m, 2 H, H-4′ and 5-CH₂b), 3.85(AB dd, J=11.4 and 3.3 Hz, 1 H, H-5′a), 3.74 (AB dd, J=11.4 and 2.8 Hz,1 H, H-5′b), 2.90 (sep, J=6.9 Hz, 1 H, OSO₂Ph(p-CH(CH₃)₂)), 2.53 (m, 1H, H-2′a), 1.94 (m, 1 H, H-2′b), 1.29 (d, J=6.9 Hz, 6 H,OSO₂Ph(p-CH(CH₃)₂)), 1.26 (d, J=7.2 Hz, 12 H, OSO₂Ph(o-CH(CH₃)₂)₂), 0.87(s, 18 H, (CH₃)₃CSi), 0.85 (s, 9 H, (CH₃)₃CC), 0.06 and 0.05 (2 s, 12 H,2 (CH₃)₂Si); ¹³C NMR (100 MHz, CDCl₃): δ 166.01 (C), 154.43 (C), 153.54(C), 151.21 (C), 151.08 (C), 145.07 (CH), 141.49 (CH), 133.16 (C),132.32 (CH), 131.76 (CH), 131.15 (C), 124.08 (CH), 104.64 (C), 92.25(C), 88.60 (CH), 87.83 (CH), 82.48 (CH), 71.83 (CH), 64.33 (CH₂), 62.75(CH₂), 42.31 (CH₂), 36.51 (C), 34.26 (CH), 29.68 (CH), 25.91 (C(CH₃)₃),25.73 (C(CH₃)₃), 24.51 (C(CH₃)₃), 23.52 (CH₃), 23.45 (CH₃), 18.37 (C),17.98 (C), −4.57 (CH₃), −4.91 (CH₃), −5.34 (CH₃), −5.39 (CH₃).

3′,5′-O-Bis-(tert-butyldimethylsilyl)-5-[(R orS)-1-(4-iodo-2-nitrophenyl)-2,2-dimethyl-propyloxy]methyl-2′-deoxcytidine(dC.z3): Ammonia (0.5 M in dioxane, 4 mL, 2.0 mmol) was added tocompound dU.z2 (169 mg, 0.158 mmol) and the mixture was transferred intoa sealed tube, and heated at 96° C. for 16 hours. After cooled to roomtemperature, the mixture was concentrated under reduced pressure andpurified by silica gel column chromatography to afford3′,5′-O-bis-(tert-butyldimethylsilyl)-5-[(R orS)-1-(4-iodo-2-nitrophenyl)-2,2-dimethyl-propyloxy]methyl-2′-deoxcytidinedC.z3 (83 mg, 65%). ¹H NMR (400 MHz, CDCl₃) δ 8.04 (d, 1 H, J=1.6 Hz,Ph-H), 7.92 (dd, J=8.3 and 1.6 Hz, 1 H, Ph-H), 7.66 (s, 1 H, H-6), 7.42(d, 1 H, J=8.3 Hz, Ph-H), 6.29 (t, 1 H, J=6.7 Hz, H-1′), 4.81 (s, 1 H,PhCH), 4.32 (m, 1 H, H-3′), 4.28 (AB d, J=12.9 Hz, 1 H, 5-CH₂a), 4.04(AB d, J=12.9 Hz, 1 H, 5-CH₂b), 3.96 (m, 1 H, H-4′), 3.79 (AB dd, J=11.2and 3.2 Hz, 1 H, H-5′a), 3.72 (AB dd, J=11.2 and 2.8 Hz, 1 H, H-5′b),2.42 (m, 1 H, H-2′a), 1.90 (m, 1 H, H-2′b), 0.88 (s, 9 H, (CH₃)₃CSi),0.81 (s, 9 H, (CH₃)₃CSi), 0.80 (s, 9 H, (CH₃)₃CC), 0.07 (s, 3 H, CH₃Si),0.06 (s, 3 H, CH₃Si), −0.05 (s, 6 H, (CH₃)₂Si); ¹³C NMR (100 MHz,CDCl₃): δ 165.23 (C), 155.63 (C), 151.24 (C), 141.01 (CH), 140.65 (CH),133.29 (C), 132.26 (CH), 131.35 (CH), 101.59 (C), 92.38 (C), 87.80 (CH),86.29 (CH), 81.00 (CH), 72.17 (CH), 66.80 (CH₂), 62.92 (CH₂), 42.10(CH₂), 36.27 (C), 25.82 (C(CH₃)₃), 25.80 (C(CH₃)₃), 25.77 (C(CH₃)₃),18.27 (C), 17.99 (C), −4.58 (CH₃), −4.85 (CH₃), −5.47 (CH₃), −5.53(CH₃).

5-[(R orS)-1-(4-Iodo-2-nitrophenyl)-2,2-dimethyl-propyloxy]methyl-2′-deoxy-cytidine(dC.z4): A solution of tetra-n-butylammonium fluoride trihydrate (81 mg,0.26 mmol) in THF (2 mL) was added to a solution of compound dC.z3 (82mg, 0.10 mmol) in THF (3 mL). The mixture was stirred at roomtemperature for 1 hour, then concentrated in vacuo and purified bysilica gel column chromatography to give 5-[(R orS)-1-(4-iodo-2-nitrophenyl)-2,2-dimethyl-propyloxy]methyl-2′-deoxcytidinedC.z4 (43 mg, 73%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.23 (d, 1 H, J=1.6 Hz,Ph-H), 8.06 (dd, J=8.4 and 1.6 Hz, 1 H, Ph-H), 7.73 (s, 1 H, H-6), 7.38(d, 1 H, J=8.4 Hz, Ph-H), 7.37 and 6.64 (2 br. s, 2 H, D₂O exchangeable,4-NH₂), 6.10 (t, 1 H, J=6.8 Hz, H-1′), 5.20 (d, J=4.0 Hz, 1 H, D₂Oexchangeable, 3′-OH), 4.88 (t, J=5.2 Hz, 1 H, D₂O exchangeable, 5′-OH),4.76 (s, 1 H, PhCH), 4.19 (m, 1 H, H-3′), 4.10 (s, 1 H, 5-CH₂a and5-CH₂b), 3.75 (m, 1 H, H-4′), 3.51 (m, 2 H, H-5′a and H-5′b), 2.08 (m, 1H, H-2′a), 1.93 (m, 1 H, H-2′b), 0.76 (s, 9 H, (CH₃)₃C).

5-[(R orS)-1-(4-{3-Trifluoroacetamido-1-propynyl}-2-nitrophenyl)-2,2-dimethyl-propyloxy]methyl-2′-deoxcytidine(dC.z5): A mixture of compound dC.z4 (40 mg, 0.069 mmol),N-propargyltrifluoroacetylamide (32 mg, 0.208 mmol), CuI (3 mg, 0.014mmol), Et₃N (0.01 mL, 0.138 mmol) andtetrakis(triphenylphosphine)palladium(0) (8 mg, 0.007 mmol) in anhydrousDMF (3 mL) was stirred at room temperature for 4.5 hours. Methanol (3mL) and methylene chloride (3 mL) were added, followed by sodiumbicarbonate (32 mg, 0.380 mmol). The mixture was stirred for 15 minutes,then concentrated in vacuo and purified by silica gel columnchromatography to yield 5-[(R orS)-1-(4-{3-trifluoroacetamido-1-propynyl}-2-nitrophenyl)-2,2-dimethyl-propyl-oxy]methyl-2′-deoxycytidinedC.z5 (35 mg, 85%). ¹H NMR (400 MHz, DMSO-d₆): δ 10.13 (br t, 1 H, D₂Oexchangeable, NHTFA), 7.94 (d, 1 H, J=1.6 Hz, Ph-H), 7.75 (m, 2 H, Ph-Hand H-6), 7.63 (d, 1 H, J=8.2 Hz, Ph-H), 7.34 and 6.66 (2 br. s, 2 H,D₂O exchangeable, 4-NH₂), 6.10 (t, 1 H, J=6.8 Hz, H-1′), 5.20 (d, J=4.2Hz, 1 H, D₂O exchangeable, 3′-OH), 4.89 (t, J=5.4 Hz, 1 H, D₂Oexchangeable, 5′-OH), 4.82 (s, 1 H, PhCH), 4.31 (d, 2 H, J=5.4 Hz, CH₂NHTFA), 4.19 (m, 1 H, H-3′), 4.14 (AB d, J=12.5 Hz, 1 H, 5-CH₂a), 4.10(AB d, J=12.5 Hz, 1 H, 5-CH₂b), 3.75 (m, 1 H, H-4′), 3.51 (m, 2 H, H-5′aand H-5′b), 2.08 (m, 1 H, H-2′a), 1.93 (m, 1 H, H-2′b), 0.76 (s, 9 H,(CH₃)₃C); ¹³C NMR (100 MHz, CD₃OD): δ 164.83 (C), 157.51 (C), 157.14(C), 156.45 (C), 150.34 (C), 141.65 (CH), 134.48 (CH), 133.91 (C),130.26 (CH), 126.50 (CH), 123.01 (C), 87.55 (CH), 86.28 (CH), 86.08 (C),80.77 (CH), 80.17 (C), 70.623 (CH), 65.74 (CH₂), 61.33 (CH₂), 40.51(CH₂), 36.19 (C), 29.09 (CH₂), 24.83 (CH₃)₃C).

5-[(R orS)-1-(4-{3-Amino-1-propynyl}-2-nitrophenyl)-2,2-dimethyl-propyl-oxy]methyl-2′-deoxcytidine-5′-triphosphate(dC.z6): POCl₃ (2.5 μL, 0.027 mmol) was added to a solution of compounddC.z5 (11 mg, 0.018 mmol) and proton sponge (8 mg, 0.036 mmol) intrimethylphosphate (0.3 mL) at 0° C. and stirred for two hours.Additional POCl₃ (2.5 μL, 0.027 mmol) was added twice in one hourinterval. A solution of bis-tri-n-butylammonium pyrophosphate (43 mg,0.09 mmol) and tri-n-butylamine (20 μL) in anhydrous DMF (0.2 mL) wasadded. After five minutes of stirring, triethylammonium bicarbonatebuffer (1 M, pH 7.5; 5 mL) was added. The reaction was stirred for onehour at room temperature and then concentrated in vacuo at 25° C. Theresidue was dissolved in water (5 mL), filtered, and part of the mixturewas purified with reverse-phase HPLC using a Perkin Elmer OD-300 C₁₈column (4.6×250 mm) to yield 5-{(R orS)-1-[4-(3-trifluoroacetamido-1-propynyl)-2-nitrophenyl]-2,2-dimethyl-propyloxy}methyl-2′-deoxy-cytidine-5′-trihosphate.Mobile phase: A, 100 mM triethylammonium acetate (TEAA) in water (pH7.0); B, 100 mM TEAA in water/CH₃CN (30:70). The purified triphosphatewas then treated with concentrated ammonium hydroxide (27%) at roomtemperature for two hours to yield 5-{(R orS)-1-[4-(3-amino-1-propynyl)-2-nitrophenyl]-2,2-dimethyl-propyloxy}methyl-2′-deoxycytidine-5′-trihosphatedC.z6. ¹H NMR (400 MHz, CD₃OD) δ 8.01 (s, 1 H, H-6), 7.84 (d, 1 H, J=8.0Hz, Ph-H), 7.67 (d, 1 H, J=8.0 Hz, Ph-H), 7.51 (m, 1 H, Ph-H), 6.11 (t,1 H, J=6.4 Hz, H-1′), 4.54 (AB d, 1 H, J=13.6 Hz, 5-CH₂a), 4.49 (m, 1 H,H-3′), 4.35 (d, 1 H, J=13.6 Hz, 5-CH₂b), 4.15-3.81 (m, 4 H, H-4′, H-5′and CH₂), 2.31 (m, 1 H, H-2′a), 2.12 (m, 1 H, H-2′b), 0.81 (s, 9 H,(CH₃)₃C); ³¹P NMR (162 MHz, D₂O) for diastereomers: 6-5.20 (d, J=19.4Hz), −10.77 (d, J=19.4 Hz), −20.96 (t, J=19.4 Hz).

6-TAMRA labeled 5-[(R orS)-1-(4-{3-amino-1-propynyl}-2-nitrophenyl)-2,2-dimethyl-propyloxy]methyl-2′-deoxcytidine-5′-triphosphate(WW3p091): A solution of Cy5 mono NHS (0.65 mg, 1.23 μmol) in anhydrousDMSO (26 μL) was added to a solution of triphosphate dC.z6 (0.386 μmol)in Na₂CO₃/NaHCO₃ buffer (0.1 M, pH 9.2; 200 μL) and left at roomtemperature for one hour. The reaction was purified by reverse-phaseHPLC using a Perkin Elmer OD-300 C₁₈ column (4.6×250 mm) to yield the6-TAMRA labeled triphosphate WW3p091. Mobile phase: A, 100 mM TEAA inwater (pH 7.0); B, 100 mM TEAA in water/CH₃CN (30:70).

Example 7 Synthesis of 7-Deazaguanosine Analogs Synthesis of7-(2-nitrobenzyloxy)methyl-7-deaza-2′-deoxyguanosine-5′-triphosphate

6-Chloro-2-(trifluoroacetyl)amino-7-deazapurine (dG.22): Compound dG.22was synthesized according to the procedure described by Seela and Peng(2006, which is incorporated herein by reference). To a solution of2-amino-6-chloro-7-deazapurine (2.00 g, 11.86 mmol) in anhydrouspyridine (15 mL) was added trifluoroacetic anhydride (2.18 mL, 15.54mmol) over fifteen minutes. The solution was stirred at room temperaturefor three hours and concentrated in vacuo and co-evaporated with water(2 mL) two times. The material was then filtered, washed with coldwater, and dried over KOH under vacuum to yield6-choloro-2-(trifluoroacetyl)amino-7-deazapurine dG.22 (2.86 g, 91%) asan amber solid.

2-Amino-6-chloro-7-iodo-7-deazapurine (dG.23): Compound dG.23 wassynthesized according to the procedure described by Seela and Peng(2006, which is incorporated herein by reference). To a suspension ofcompound dG.22 (2.86 g, 10.81 mmol) in anhydrous CH₂Cl₂ (51 mL) wasadded N-iodosuccinimide (2.68 g, 11.89 mmol). The mixture was protectedfrom light while stirring at room temperature for two hours. Thereaction was then diluted with 322 mL CH₂Cl₂ and filtered; theprecipitate was then dissolved in 7N NH₃ in methanol solution (41 mL)and stirred at room temperature for three hours. The resulting solid wasfiltered and dried in vacuo to yield2-amino-6-chloro-7-iodo-7-deazapurine dG.23 (1.86 g, 59%) as an ambersolid.

2-Amino-6-chloro-9-[β-D-3′,5′-O-di-(p-toluoyl)-2′-deoxyribofuranosyl]-7-iodo-7-deazapurine(dG.24): To a suspension of KOH (1.38 g, 22.16 mmol) andtris(3,6-dioxaheptyl)amine (0.26 mL, 0.80 mmol) in anhydrousacetonitrile (76 mL) was added compound dG.23 (1.86 g, 6.33 mmol). Afterstirring the mixture for five minutes,2-deoxy-3,5-di-O-(p-toluoyl)-α-D-ribofuranosyl chloride (3.20 g, 8.23mmol) was added over 15 minutes. The reaction was stirred at roomtemperature for 30 minutes then filtered, and the precipitate was washedwith acetonitrile (75 mL). The combined filtrated was concentrated invacuo and purified by silica gel chromatography to yield2-amino-6-chloro-9-[β-D-3′,5′-O-di-(p-toluoyl)-2′-deoxyribofuranosyl]-7-iodo-7-deazapurinedG.24 (3.29 g, 80%) as a white foam. ¹H NMR (400 MHz, CDCl₃): δ 8.05 (m,4 H, Ph-H), 7.39 (s, 1 H, H-8), 7.37 (m, 4 H, Ph-H), 6.66 (dd, 1 H,J=8.0 and 6.0 Hz, H-1′), 5.83 (m, 1 H, H-3′), 5.24 (bs, 2 H, 2-NH₂),4.85 (dd, 1 H, H-5′a), 4.74 (dd, 1 H, H-5′b), 4.68 (m, 1 H, H-4′), 2.88(m, 1 H, H-2′a), 2.76 (m, 1 H, H-2′b), 2.54 (s, 3 H, Ph-CH₃), 2.53 (s, 3H, Ph-CH₃).

2-Amino-6-chloro-9-(β-D-2′-deoxyribofuranosyl)-7-iodo-7-deazapurine(dG.25): Compound dG.24 (3.29 g, 5.09 mmol) was dissolved in 7N NH₃ inmethanol solution (153 mL) and stirred at room temperature for 32 hours.The mixture was concentrated in vacuo and purified by silica gelchromatography to yield2-amino-6-chloro-9-(β-D-2′-deoxyribofuranosyl)-7-iodo-7-deazapurinedG.25 (1.66 g, 80% yield) as a white foam. ¹H NMR (400 MHz, DMSO-d₆): δ7.60 (s, 1 H, H-8), 6.87 (bs, 2 H, 2-NH₂), 6.40 (dd, 1 H, J=8.0 and 6.0Hz, H-1′), 5.25 (d, 1 H, 3′-OH), 4.93 (t, 1 H, 5′-OH), 4.30 (m, 1 H,H-3′), 3.78 (m, 1 H, H-4′), 3.51 (m, 2 H, H-5′a and H-5′b), 2.40 (m, 1H, H-2′a), 2.13 (m, 1 H, H-2′b).

9-[β-D-3′,5′-O-Bis-(tert-butyldimethylsilyl)-2′-deoxyribofuranosyl]-2-(tert-butyldimethylsilyl)amino-6-chloro-7-iodo-7-deazapurine(dG.26): Compound dG.25 (0.29 g, 0.70 mmol) was evaporated fromanhydrous pyridine three times (3 mL each) and then dissolved inanhydrous DMF (5 mL). tert-Butyldimethylsilyl chloride (1.27 g, 8.43mmol) and imidazole (1.15 g, 16.86 mmol) were added, and the mixture wasstirred at 40° C. for 42 hours (additional tert-butyldimethylsilylchloride (0.64 g, 4.22 mmol) and imidazole (0.57 g, 8.43 mmol were addedevery six hours). The reaction was concentrated in vacuo and purified bysilica gel chromatography to yield9-[β-D-3′,5′-O-bis-(tert-butyldimethylsilyl)-2′-deoxyribofuranosyl]-2-(tert-butyldimethylsilyl)amino-6-chloro-7-iodo-7-deazapurinedG.26 (0.30 g, 56% yield) as a white foam. ¹H NMR (400 MHz, CDCl₃): δ7.35 (s, 1 H, H-8), 6.53 (t, 1 H, J=6.0 Hz, H-1′), 4.70 (s, 1 H, 2-NH),4.47 (m, 1 H, H-3′), 3.97 (m, 1 H, H-4′), 3.78 (m, 2 H, H-5′a andH-5′b), 2.23 (m, 2 H, H-2′a and H-2′b), 0.98 (s, 9 H, (CH₃)₃CSi), 0.95(s, 9 H, (CH₃)₃CSi), 0.90 (s, 9 H, (CH₃)₃CSi), 0.29 (2 s, 6 H,(CH₃)₂Si), 0.13 (2 s, 6 H, CH₃)₂Si), 0.09 (s, 6 H, CH₃)₂Si).

9-[β-D-3′,5′-O-Bis-(tert-butyldimethylsilyl)-2′-deoxyribofuranosyl]-2-(tert-butyl-dimethylsilyl)amino-6-chloro-7-methoxycarbonyl-7-deazapurine(dG.27): A solution of dG.26 (105 mg, 0.139 mmol) was dissolved inanhydrous 1,4-dioxane (6 mL). Anhydrous methanol (6 mL) andtriethylamine (0.04 mL) were added, and the mixture was stirred for tenminutes under carbon monoxide atmosphere, followed by addition ofbis(benzonitrile)dichloropalladium(II). The reaction was stirred at 50°C. for 48 hours under CO atmosphere, and then concentrated in vacuo. Theresidue was purified by silica gel chromatography to yield9-[β-D-3′,5′-O-bis-(tert-butyldimethylsilyl)-2′-deoxyribofuranosyl]-2-(tert-butyldimethylsilyl)amino-6-chloro-7-methoxycarbonyl-7-deazapurinedG.27 (112 mg, 90%) as a viscous oil. ¹H NMR (400 MHz, CDCl₃): δ 7.92(s, 1 H, H-8), 6.57 (dd, 1 H, J=8.0 and 6.0 Hz, H-1′), 4.78 (s, 1 H,2-NH), 4.49 (m, 1 H, H-3′), 4.02 (m, 1 H, H-4′), 3.85 (s, 3 H, CH₃),3.81 (m, 2 H, H-5′a and H-5′b), 2.25 (m, 2 H, H-2′a and H-2′b), 0.98 (s,9 H, (CH₃)₃CSi), 0.93 (s, 9 H, (CH₃)₃CSi), 0.92 (s, 9 H, (CH₃)₃CSi),0.31 (s, 6 H, (CH₃)₂Si), 0.13 (2 s, 6 H, (CH₃)₂Si), 0.11 (s, 6 H,(CH₃)₂Si); ¹³C NMR (100 MHz, CDCl₃): δ 163.04 (C), 160.06 (C), 154.46(C), 153.04 (C), 129.81 (CH), 107.79 (C), 107.51 (C), 87.86 (CH), 84.05(CH), 72.73 (CH), 63.21 (CH₂), 51.26 (CH₃), 42.21 (CH₂), 26.48 (CH₃),25.96 (CH₃), 25.72 (CH₃), 18.42 (C), 18.03 (C), 17.59 (C), −4.73 (CH₃),−4.80 (CH₃), −5.49 (CH₃), −5.57 (CH₃).

9-[β-D-3′,5′-O-Bis-(tert-butyldimethylsilyl)-2′-deoxyribofuranosyl]-2-(tert-butyldimethylsilyl)amino-6-chloro-7-hydroxymethyl-7-deazapurine(dG.28): To a solution of dG.27 (52 mg, 0.076 mmol) in anhydrous THF (3mL) lithium borohydride (0.007 g, 0.305 mmol) was added, followed bymethanol (0.05 mL). The reaction mixture was heated at reflux for onehour. Upon cooling down, the reaction mixture was diluted withdichloromethane (100 mL), quenched with water (10 mL); the organic layerwas separated, washed two times with brine (10 mL each), dried overanhydrous sodium sulfate, and concentrated in vacuo. The residue waspurified by silica gel chromatography to yield9-[β-D-3′,5′-O-bis-(tert-butyldimethylsilyl)-2′-deoxyribo-furanosyl]-2-(tert-butyldimethylsilyl)amino-6-chloro-7-hydroxymethyl-7-deazapurinedG.28 (0.12 g, 45%) as a viscous oil. ¹H NMR (400 MHz, CDCl₃): δ 7.16(s, 1 H, H-8), 6.56 (t, 1 H, J=6.4 Hz, H-1′), 4.79 (AB d, J=13.6 Hz,7-CH₂a), 4.75 (AB d, J=13.6 Hz, 7-CH₂b), 4.70 (s, 1 H, 2-NH), 4.50 (m, 1H, H-3′), 3.96 (m, 1 H, H-4′), 3.76 (m, 2 H, H-5′a and H-5′b), 2.23 (m,2 H, H-2′a and H-2′b), 0.98 (s, 9 H, (CH₃)₃CSi), 0.94 (s, 9 H,(CH₃)₃CSi), 0.92 (s, 9 H, (CH₃)₃CSi), 0.30 (s, 3 H, (CH₃)₂Si), 0.29 (s,3 H, (CH₃)₂Si), 0.11 (s, 6 H, (CH₃)₂Si), 0.10 (s, 6 H, (CH₃)₂Si); ¹³CNMR (100 MHz, CDCl₃): δ 160.07 (C), 154.29 (C), 151.23 (C), 120.92 (CH),115.65 (C), 108.44 (C), 87.26 (CH), 83.21 (CH), 72.50 (CH), 63.28 (CH₂),57.15 (CH₂), 42.33 (CH₂), 26.55 (CH₃), 25.97 (CH₃), 25.73 (CH₃), 18.42(C), 17.93 (C), 17.61 (C), −4.71 (CH₃), −4.75 (CH₃), −5.34 (CH₃), −5.47(CH₃).

9-[β-D-3′,5′-O-Bis-(tert-butyldimethylsilyl)-2′-deoxyribofuranosyl]-2-(tert-butyl-dimethylsilyl)amino-6-chloro-7-(2-nitrobenzyloxy)methyl-7-deazapurine(dG.29): To a solution of compound dG.28 (150 mg, 0.23 mmol) in CH₂Cl₂(3 mL) were added n-Bu₄NBr (37 mg, 0.12 mmol), 2-nitrobenzyl bromide(148 mg, 0.68 mmol) and 1 M NaOH solution (3 mL). The reaction mixturewas stirred vigorously at room temperature for two days in the dark. Theorganic layer was separated, dried over Na₂SO₄, concentrated in vacuo,and purified by silica gel chromatography to yield9-[β-D-3′,5′-O-bis-(tert-butyldimethylsilyl)-2′-deoxyribofuranosyl]-2-(tert-butyldimethylsilyl)amino-6-chloro-7-(2-nitrobenzyloxy)methyl-7-deazapurinedG.29 (87 mg, 48%) as a viscous oil. ¹H NMR (400 MHz, CDCl₃): δ 8.06(dd, 1 H, J=8.0 and 1.2 Hz, Ph-H), 7.87 (d, 1 H, J=7.2 Hz, Ph-H), 7.61(dt, 1 H, J=7.6 and 1.2 Hz, Ph-H), 7.43 (m, 1 H, Ph-H), 7.20 (s, 1 H,H-8), 6.56 (dd, 1 H, J=7.6 and 6.0 Hz, H-1′), 4.99 (s, 2 H, PhCH₂), 4.83(AB d, 1 H, J=11.4 Hz, 7-CH₂a), 4.75 (AB d, 1 H, J=11.4 Hz, 7-CH₂b),4.67 (s, 1 H, 2-NH), 4.50 (m, 1 H, H-3′), 3.96 (m, 1 H, H-4′), 3.77 (m,2 H, H-5′a and H-5′b), 2.25 (m, 2 H, H-2′a and H-2′b), 0.98 (s, 9 H,(CH₃)₃CSi), 0.92 (s, 18 H, (CH₃)₃CSi), 0.30 (s, 3 H, (CH₃)₂Si), 0.29 (s,3 H, (CH₃)₂Si), 0.09 (m, 12 H, (CH₃)₂Si); ¹³C NMR (100 MHz, CDCl₃): δ159.94 (C), 154.18 (C), 151.78 (C), 147.16 (C), 135.23 (C), 133.6 (CH),129.0 (CH), 127.75 (CH), 124.49 (CH), 121.85 (CH), 112.17 (C), 108.74(C), 87.24 (CH), 83.22 (CH), 72.50 (CH), 68.48 (CH₂), 65.04 (CH₂), 63.27(CH₂), 41.31 (CH₂), 26.52 (CH₃), 25.93 (CH₃), 25.7 (CH₃), 18.36 (C),17.89 (C), 17.56 (C), −4.75 (CH₃), −4.81 (CH₃), −5.39 (CH₃), −5.52(CH₃).

2-Amino-6-chloro-9-[β-D-2′-deoxyribofuranosyl]-7-(2-nitrobenzyloxy)methyl-7-deazapurine(dG.30): A solution of n-Bu₄NF (123 mg, 0.39 mmol) in THF (2 mL) wasadded dropwise to a solution of compound dG.29 (105 mg, 0.13 mmol) inTHF (3 mL) at 0° C. The reaction mixture was stirred at 0° C. for onehour and then at room temperature for two hours. The reaction wasconcentrated in vacuo and purified by silica gel chromatography to yield2-amino-6-chloro-9-[β-D-2′-deoxyribofuranosyl]-7-(2-nitrobenzyloxy)methyl-7-deazapurinedG.30 (57 mg, 95%) as a yellow foam. ¹H NMR (400 MHz, DMSO-d₆): 6, 8.02(m, 1 H, Ph-H), 7.74 (m, 2 H, Ph-H), 7.55 (m, 1 H, Ph-H), 7.41 (s, 1 H,H-8), 6.73 (s, 2 H, D₂O exchangeable, NH₂), 6.41 (dd, 1 H, J=8.4 and 6.0Hz, H-1′), 5.26 (d, 1 H, D₂O exchangeable, 3′-OH), 4.91 (t, 1 H, D₂Oexchangeable, 5′-OH), 4.88 (s, 2 H, Ph-CH₂), 4.66 (dd, 2 H, J=11.6 Hz,7-CH₂), 4.31 (m, 1 H, H-3′), 3.78 (m, 1 H, H-4′), 3.50 (m, 2 H, H-5′),2.38 (m, 1 H, H-2′a), 2.15 (m, 1 H, H-2′b).

7-(2-Nitrobenzyloxy)methyl-7-deaza-2′-deoxyguanosine (dG.31): A mixtureof dG.29 (38 mg, 0.084 mmol) and 1,4-diazabicyclo[2.2.2]octane (11 mg,0.1 mmol) in water (4 mL) was heated to reflux under a nitrogenatmosphere for 4 hours. Water was removed in vacuo, and the residue wasevaporated from methanol three times (3 mL each), and purified by silicagel chromatography to yield7-(2-nitrobenzyloxy)methyl-7-deaza-2′-deoxyguanosine dG.31 (11 mg, 30%).¹H NMR (400 MHz, DMSO-d₆): δ 10.4 (s, 1 H, D₂O exchangeable, N—H), 8.03(dd, 1 H, J=8.4 and 0.8 Hz, Ph-H), 7.83 (d, 1 H, J=7.6 Hz, Ph-H), 7.73(m, 1 H, Ph-H), 7.55 (m, 1 H, Ph-H), 6.92 (s, 1 H, H-8), 6.28 (m, 1 H,H-1′), 6.26 (bs, 2 H, D₂O exchangeable, NH₂), 5.21 (d, 1 H, D₂Oexchangeable, 3′-OH), 4.89 (t, 1 H, D₂O exchangeable, 5′-OH), 4.88 (s, 2H, Ph-CH₂), 4.60 (dd, 2 H, 7-CH₂), 4.28 (m, 1 H, H-3′), 3.74 (m, 1 H,H-4′), 3.48 (m, 2 H, H-5′), 2.32 (m, 1 H, H-2′a), 2.08 (m, 1 H, H-2′b).

7-(2-Nitrobenzyloxy)methyl-7-deaza-2′-deoxyguanosine-5′-triphosphate(WW5p107): POCl₃ (5 μL, 0.05 mmol) was added to a solution of compounddG.31 (11 mg, 0.025 mmol) in trimethylphosphate (0.3 mL), and thereaction was stirred at 0° C. under a nitrogen atmosphere for two hours.A solution of bis-tri-n-butylammonium pyrophosphate (118 mg, 0.25 mmol)and tri-n-butylamine (50 μL) in anhydrous DMF (0.5 mL) was added. After30 minutes of stirring, triethylammonium bicarbonate buffer (1 M, pH7.5; 5 mL) was added. The reaction was stirred for one hour at roomtemperature and then concentrated in vacuo. The residue was dissolved inwater (10 mL), filtered, and purified by anion exchange chromatographyusing a Q Sepharose FF column (2.5×10 cm) with a linear gradient of 25%acetonitrile/75% triethylammonium bicarbonate (TEAB, 0.1M) to 25%acetonitrile/75% TEAB (1.5 M) over 240 min at 4.5 ml/min. The fractionscontaining triphosphate were combined and lyophilized to give7-(2-nitrobenzyloxy)methyl-7-deaza-2′-deoxyguanosine-5′-triphosphateWW5p107 which was further purified by reverse phase HPLC on a PerkinElmer Aquapore OD-300 column (7 μm, 250×4.6 mm). Mobile phase: A, 100 mMtriethylammonium acetate (TEAA) in water; B, 100 mM TEAA in water/CH₃CN(30:70).

Synthesis of7-[1-(2-nitrophenyl)-2-methyl-propyloxy]methyl-7-deaza-2′-deoxyguanosine-5′-triphosphate

9-[β-D-3′,5′-O-Bis-(tert-butyldimethylsilyl)-2′-deoxyribofuranosyl]-2-(tert-butyldimethylsilyl)amino-6-chloro-7-chloromethyl-7-deazapurine(dG.32): To a solution of dG.27 (1.22 g, 1.84 mmol) in carbontetrachloride (24 mL, freshly distilled from CaH₂) were added potassiumcarbonate (1.00 g, 7.36 mmol) and triphenyl phosphine (1.20 g, 4.60mmol). The reaction was stirred at reflux for 24 hours. The mixture wasconcentrated in vacuo and purified by silica gel chromatography to yield9-[β-D-3′,5′-β-bis-(tert-butyldimethylsilyl)-2′-deoxyribofuranosyl]-2-(tert-butyldimethylsilyl)amino-6-chloro-7-chloromethyl-7-deazapurinedG.32 (0.54 g, 43%) as foam. ¹H NMR (400 MHz, CDCl₃): δ 7.16 (s, 1 H,H-8), 6.54 (dd, 1 H, J=8.0 and 6.0 Hz, H-1′), 4.78 (AB d, J=11.4 Hz,7-CH₂a), 4.68 (AB d, J=11.4 Hz, 7-CH₂b), 4.64 (s, 1 H, 2-NH), 4.47 (m, 1H, H-3′), 3.94 (m, 1 H, H-4′), 3.73 (m, 2 H, H-5′a and H-5′b), 2.20 (m,2 H, H-2′a and H-2′b), 0.98 (s, 9 H, (CH₃)₃CSi), 0.91 (s, 9 H,(CH₃)₃CSi), 0.90 (s, 9 H, (CH₃)₃CSi), 0.28 (2 s, 6 H, (CH₃)₂Si), 0.09 (2s, 6 H, (CH₃)₂Si), 0.07 (2 s, 6 H, (CH₃)₂Si); ¹³C NMR (100 MHz, CDCl₃):δ 159.85 (C), 154.16 (C), 151.82 (C), 121.86 (CH), 112.80 (C), 108.94(C), 87.23 (CH), 83.17 (CH), 72.58 (CH), 63.35 (CH₂), 41.24 (CH₂), 29.72(CH₂), 26.57 (CH₃), 25.97 (CH₃), 25.74 (CH₃), 18.38 (C), 17.93 (C),17.62 (C), −4.71 (CH₃), −4.74 (CH₃), −4.76 (CH₃), −5.37 (CH₃), −5.49(CH₃).

2-Amino-6-chloro-9-[β-D-2′-deoxyribofuranosyl]-7-[1-(2-nitrophenyl)-2-methyl-propyl-oxy]methyl-7-deazapurine(dG.33): Compound dG.32 (0.82 g, 1.21 mmol) and1-(2-nitrophenyl)-2-methyl-propanol (2.36 g, 12.10 mmol) were dissolvedin anhydrous dichloromethane (10 mL). The solvent was removed in vacuo,and the residue was heated in vacuo at 124° C. for 22 hours, thendissolved in ethyl acetate and purified by silica gel chromatography toyield crude9-[β-D-3′,5′-O-bis-(tert-butyldimethylsilyl)-2′-deoxyribofuranosyl]-2-amino-6-chloro-7-[1-(2-nitrophenyl)-2-methyl-propyloxy]methyl-7-deazapurine.This intermediate was then dissolved in tetrahydrofuran (14 mL) andtreated with tetra-n-butylammonium fluoride trihydrate (0.954 g, 3.03mmol). After 30 minutes, the mixture was evaporated and purified bycolumn chromatography to yield9-[β-D-2′-deoxyribofuranosyl]-2-amino-6-chloro-7-[1-(2-nitrophenyl)-2-methyl-propyl-oxy]methyl-7-deazapurinedG.33 (41 mg, 7%, 1:1 mixture of diastereomers). ¹H NMR (400 MHz, CD₃OD)for diastereomers: δ 7.82 and 7.79 (2 dd, J=8.0 and 1.2 Hz, 1 H, Ph-H),7.72 (dt, J=8.0 and 1.6 Hz, 1 H, Ph-H), 7.60 (m, 1 H, Ph-H), 7.41 (m, 1H, Ph-H), 7.17 and 7.14 (2 s, 1 H, H-8), 6.41 (m, 1 H, H-1′), 4.71 (t, 1H, J=6.8 Hz Ph-CH), 4.48 (m, 2 H, 7-CH₂ and H-3′), 3.94 (m, 1 H, H-4′),3.71 (m, 2 H, H-5′), 2.53 (m, 1 H, H-2′a), 2.27 (m, 1 H, H-2′b), 1.92(oct, J=6.8 Hz, 1 H, CHCH(CH₃)₂), 0.96 and 0.94 (2 d, J=6.8 Hz, 3 H,CH₃), 0.80 and 0.76 (2 d, J=6.8 Hz, 3 H, CH₃); ¹³C NMR (100 MHz, CD₃OD)for diastereomers: δ 160.65 (C), 155.83 and 155.78 (C), 153.54 and153.45 (C), 151.39 and 151.12 (C), 138.42 and 138.27 (C), 133.89 and133.77 (CH), 130.66 and 130.56 (CH), 129.44 and 129.36 (CH), 125.66 and125.29 (CH), 124.97 and 124.88 (CH), 113.67 and 113.39 (C), 110.08 (C),88.81 and 88.78 (CH), 85.52 and 85.27 (CH), 81.60 and 81.87 (CH), 73.08(CH), 64.71 and 64.18 (CH₂), 63.84 and 63.78 (CH₂), 41.00 and 40.86(CH₂), 36.31 and 36.28 (CH), 19.77 and 19.73 (CH₃), 18.58 and 18.52(CH₃).

7-[1-(2-nitrophenyl)-2-methyl-propyloxy]methyl-7-deaza-2′-deoxyguanosine(dG.34): A mixture of dG.33 (54 mg, 0.11 mmol) and1,4-diazabicyclo[2.2.2]octane (25 mg, 0.22 mmol) in water (5 mL) washeated to reflux under a nitrogen atmosphere for three hours. Water wasremoved in vacuo, and the residue was evaporated from methanol threetimes (5 mL each), and purified by silica gel chromatography to yield7-[1-(2-nitrophenyl)-2-methyl-propyloxy]methyl-7-deaza-2′-deoxyguanosinedG.34 (15 mg, 29%, 1:1 mixture of diastereomers). ¹H NMR (400 MHz,CD₃OD) for diastereomers: δ 7.82 (m, 1 H, Ph-H), 7.76 (m, 1 H, Ph-H),7.60 (m, 1 H, Ph-H), 7.42 (m, 1 H, Ph-H), 6.81 and 6.78 (2 s, 1 H, H-8),6.28 (m, 1 H, H-1′), 4.79 (m, 1 H, Ph-CH), 4.50 (m, 3 H, 7-CH₂ andH-3′), 3.92 (m, 1 H, H-4′), 3.71 (m, 2 H, H-5′), 2.48 (m, 1 H, H-2′a),2.22 (m, 1 H, H-2′b), 1.92 (m, 1 H, CH), 0.93 (m, 3 H, CH₃), 0.83 (m, 3H, CH₃).

7-[1-(2-Nitrophenyl)-2-methyl-propyloxy]methyl-7-deaza-2′-deoxyguanosine-5′-triphosphate(WW5p143 ds1 & ds2): POCl₃ (6 μL, 0.064 mmol) was added to a solution ofcompound dG.34 (15 mg, 0.032 mmol) in trimethylphosphate (0.4 mL) andthe reaction was stirred at 0° C. under a nitrogen atmosphere for fivehours. A solution of bis-tri-n-butylammonium pyrophosphate (285 mg, 0.6mmol) and tri-n-butylamine (120 μL) in anhydrous DMF (1.2 mL) was added.After 30 minutes of stirring, triethylammonium bicarbonate buffer (1 M,pH 7.5; 10 mL) was added. The reaction was stirred for one hour at roomtemperature and then concentrated in vacuo. The residue was dissolved inwater (5 mL), filtered, and purified by anion exchange chromatographyusing a Q Sepharose FF column (2.5×10 cm) with a linear gradient of 25%acetonitrile/75% triethylammonium bicarbonate (TEAB, 0.1M) to 25%acetonitrile/75% TEAB (1.5 M) over 240 min at 4.5 ml/min. The fractionscontaining triphosphate were combined and lyophilized to give7-[1-(2-nitrophenyl)-2-methyl-propyloxy]methyl-7-deaza-2′-deoxyguanosine-5′-triphosphateWW5p143 as mixture of two diastereomers, which were separated by reversephase HPLC on a Perkin Elmer Aquapore OD-300 column (7 μm, 250×4.6 mm)to yield the single diastereomer WW5p143_ds1 (fast eluting) andWW5p143_ds2 (slow eluting). Mobile phase: A, 100 mM triethylammoniumacetate (TEAA) in water; B, 100 mM TEAA in water/CH₃CN (30:70).

Synthesis of 6-ROX labeled7-{(R)-1-[4-(3-amino-1-propynyl)-2-nitrophenyl]-2-methyl-propyloxy}methyl-7-deaza-2′-deoxyguanosine-5′-triphosphate

2-Amino-6-chloro-9-[β-D-2′-deoxyribofuranosyl]-7-[(R)-1-(4-iodo-2-nitro-phenyl)-2-methyl-propyloxy]methyl-7-deazapurine(dG.35): Compound dG.32 (0.62 g, 0.914 mmol) and(R)-1-(4-iodo-2-nitrophenyl)-2-methyl-propanol (3.52 g, 10.97 mmol) weredissolved in anhydrous dichloromethane (10 mL). The solvent was removedin vacuo, and the residue was heated in vacuo at 122° C. for 16 hours,then dissolved in ethyl acetate and purified by silica gelchromatography to yield crude2-amino-6-chloro-9-[β-D-3′,5′-O-bis-(tert-butyldimethylsilyl)-2′-deoxyribofuranosyl]-7-[(R)-1-(4-iodo-2-nitro-phenyl)-2-methyl-propyloxy]methyl-7-deazapurinethat was dissolved in tetrahydrofuran (15 mL). The intermediate wastreated with tetra-n-butylammonium fluoride trihydrate (0.72 g, 2.28mmol). After 30 minutes, the mixture was evaporated and purified bycolumn chromatography to yield2-amino-6-chloro-9-[β-D-2′-deoxyribofuranosyl]-7-[(R)-1-(4-iodo-2-nitrophenyl)-2-methyl-propyloxy]methyl-7-deazapurinedG.35 (74 mg, 13%). ¹H NMR (400 MHz, CD₃OD): δ 8.09 (d, J=1.2 Hz, 1 H,Ph-H), 7.86 (dd, J=8.0 and 1.2 Hz, 1 H, Ph-H), 7.43 (d, J=8.0 Hz, 1 H,Ph-H), 7.14 (s, 1 H, H-8), 6.38 (dd, J=8.0 and 6.0 Hz, 1 H, H-1′), 4.65(d, J=6.4 Hz, 1H, Ph-CH), 4.57 (AB d, J=12.4, 1 H, 7-CH₂a), 4.48 (m, 1H, H-3′), 4.47 (AB d, J=12.4, 1 H, 7-CH₂b), 3.95 (m, 1 H, H-4′), 3.76(AB dd, J=12.0 and 3.6 Hz, 1 H, H-5′a), 3.70 (AB dd, J=12.0 and 3.6 Hz,1 H, H-5′b), 2.52 (m, 1 H, H-2′a), 2.26 (m, 1 H, H-2′b), 1.89 (oct,J=6.8 Hz, 1 H, CHCH(CH₃)₂), 0.94 (d, J=6.8 Hz, 3 H, CH₃), 0.79 (2 d,J=6.8 Hz, 3 H, CH₃); ¹³C NMR (100 MHz, CD₃OD): δ 159.09 (C), 151.52 (C),151.95 (C), 149.53 (C), 141.20 (CH), 136.91 (C), 131.91 (CH), 130.96(CH), 124.03 (CH), 111.96 (C), 108.59 (C), 90.84 (C), 87.29 (CH), 83.97(CH), 79.92 (CH), 71.62 (CH), 63.44 (CH₂), 62.36 (CH₂), 39.31 (CH₂),34.63 (CH), 18.22 (CH₃), 16.95 (CH₃).

7-[(R)-1-(4-iodo-2-nitrophenyl)-2-methyl-propyloxy]methyl-7-deaza-2′-deoxy-guanosine(dG.36): Compound dG.35 (72 mg, 0.12 mmol) and1,4-diazabicyclo[2.2.2]octane (52 mg, 0.46 mmol) in water (5 mL) washeated to reflux under a nitrogen atmosphere for four hours. Water wasremoved in vacuo, and the residue was evaporated from methanol threetimes (5 mL each), and purified by silica gel chromatography to yield7-[(R)-1-(4-iodo-2-nitrophenyl)-2-methyl-propyloxy]methyl-7-deaza-2′-deoxyguanosinedG.36 (16 mg, 23%). ¹H NMR (400 MHz, CD₃OD): δ 8.12 (d, J=1.6 Hz, 1 H,Ph-H), 7.86 (dd, J=8.4 and 1.6 Hz, 1 H, Ph-H), 7.50 (d, J=8.4 Hz, 1 H,Ph-H), 6.80 (s, 1 H, H-8), 6.28 (dd, J=8.0 and 6.0 Hz, 1 H, H-1′), 4.74(d, J=5.6 Hz, 1 H, Ph-CH), 4.55 (AB d, J=12.0, 1 H, 7-CH₂a), 4.48 (AB d,J=12.0, 1 H, 7-CH₂b), 4.44 (m, 1 H, H-3′), 3.92 (m, 1 H, H-4′), 3.75 (ABdd, J=12.0 and 4.0 Hz, 1 H, H-5′a), 3.69 (AB dd, J=12.0 and 4.0 Hz, 1 H,H-5′b), 2.46 (m, 1 H, H-2′a), 2.23 (m, 1 H, H-2′b), 1.91 (m, 1 H, CH),0.93 (d, J=6.8 Hz, 3 H, CH₃), 0.86 (2 d, J=6.8 Hz, 3 H, CH₃).

7-{(R)-1-[4-(3-Trifluoroacetamido-1-propynyl)-2-nitrophenyl]-2-methyl-propyloxy}methyl-7-deaza-2′-deoxyguanosine(dG.37): A solution of compound dG.36 (15 mg, 0.025 mmol),N-propargyltrifluoroacetylamide (11 mg, 0.075 mmol),tetrakis(triphenylphosphine)-palladium(0) (3 mg, 0.0025 mmol), CuI (1mg, 0.005 mmol), and Et₃N (7 μL, 0.050 mmol) in anhydrous DMF (1.5 mL)was stirred at room temperature for four hours. The mixture wasconcentrated in vacuo and purified by silica gel column chromatographyto yield7-{(R)-1-[4-(3-trifluoroacetamido-1-propynyl)-2-nitrophenyl]-2-methyl-propyloxy}methyl-7-deaza-2′-deoxyguanosinedG.37 (15 mg, 99%) as a waxy solid. ¹H NMR (400 MHz, CD₃OD): δ 7.88 (d,J=1.6 Hz, 1 H, Ph-H), 7.75 (d, J=8.0 Hz, 1 H, Ph-H), 7.45 (dd, J=8.0 and1.6 Hz, 1 H, Ph-H), 6.83 (s, 1 H, H-8), 6.30 (dd, J=8.4 and 6.4 Hz, 1 H,H-1′), 4.80 (d, J=6.4 Hz, 1 H, Ph-CH), 4.56 (AB d, J=12.0, 1 H, 7-CH₂a),4.50 (AB d, J=12.0, 1 H, 7-CH₂b), 4.47 (m, 1 H, H-3′), 4.35 (s, 1 H,CH₂N), 3.94 (m, 1 H, H-4′), 3.77 (AB dd, J=12.0 and 4.0 Hz, 1 H, H-5′a),3.71 (AB dd, J=12.0 and 4.0 Hz, 1 H, H-5′b), 2.49 (m, 1 H, H-2′a), 2.23(m, 1 H, H-2′b), 1.93 (m, 1 H, CH), 0.95 (d, J=6.4 Hz, 3 H, CH₃), 0.87(d, J=6.4 Hz, 3 H, CH₃); ¹³C NMR (100 MHz, CD₃OD): δ 161.61 (C), 154.02(C), 152.67 (C), 150.49 (C), 142.69 (C), 139.77 (C), 136.37 (CH), 131.37(CH), 127.90 (CH), 123.71 (C), 119.34 (CH), 117.19 (C), 116.42 (C),88.69 (CH), 86.89 (C), 85.58 (CH), 81.93 (C), 81.42 (CH), 71.19 (CH),65.42 (CH₂), 63.97 (CH₂), 41.12 (CH₂), 36.16 (CH), 30.63 (CH₂), 19.93(CH₃), 18.00 (CH₃).

7-{(R)-1-[4-(3-Amino-1-propynyl)-2-nitrophenyl]-2-methyl-propyloxy}methyl-7-deaza-2′-deoxyguanosine-5′-triphosphate(dG.38): POCl₃ (7 μL, 0.076 mmol) was added to a solution of compounddG.37 (12 mg, 0.019 mmol) and proton sponge (8 mg, 0.038 mmol) intrimethylphosphate (0.3 mL), and the reaction was stirred at 0° C. undera nitrogen atmosphere for four hours. A solution ofbis-tri-n-butylammonium pyrophosphate (237 mg, 0.5 mmol) andtri-n-butylamine (100 μL) in anhydrous DMF (1 mL) was added. After 30minutes of stirring, triethylammonium bicarbonate buffer (1 M, pH 7.5;10 mL) was added. The reaction was stirred for one hour at roomtemperature and then concentrated in vacuo. The residue was dissolved inwater (5 mL), filtered, and purified by anion exchange chromatographyusing a Q Sepharose FF column (2.5×10 cm) with a linear gradient of 25%acetonitrile/75% triethylammonium bicarbonate (TEAB, 0.1M) to 25%acetonitrile/75% TEAB (1.5 M) over 240 min at 4.5 ml/min. The fractionscontaining triphosphate were combined and lyophilized to dryness. Theresidue was dissolved in water (5 mL) and treated with concentratedammonium hydroxide (2 mL, 27%) at room temperature for one hour to give7-{(R)-1-[4-(3-amino-1-propynyl)-2-nitrophenyl]-2-methyl-propyloxy}methyl-7-deaza-2′-deoxyguanosine-5′-triphosphatedG.38 which was purified by reverse phase HPLC on a Perkin ElmerAquapore OD-300 column (7 μm, 250×4.6 mm). Mobile phase: A, 100 mMtriethylammonium acetate (TEAA) in water; B, 100 mM TEAA in water/CH₃CN(30:70).

6-ROX labeled7-{(R)-1-[4-(3-amino-1-propynyl)-2-nitrophenyl]-2-methyl-propyloxy}methyl-7-deaza-2′-deoxyguanosine-5′-triphosphate(WW6p034): A solution of 6-ROX-SE (3.5 mg, 5.54 μmol) in anhydrous DMSO(280 μL) was added to a solution of triphosphate dG.38 (0.85 μmol) inNa₂CO₃/NaHCO₃ buffer (0.1 M, pH 9.2, 800 μL). The mixture was left atroom temperature for one hour. The dye labeled triphosphate was firstpurified by anion exchange HPLC using a Perkin Elmer AX-300 column (7μm, 250×4.6 mm). Mobile phase: A, 25% CH₃CN/75% 0.1 M TEAB; B, 25%CH₃CN/75% 1.5 M TEAB. The product was further purified by reverse-phaseHPLC using a Perkin Elmer OD-300 column (7 μm, 4.6×250 mm) to yield6-ROX labeled triphosphate WW6p034. Mobile phase: A, 100 mMtriethylammonium acetate (TEAA) in water (pH 7.0); B, 100 mM TEAA inwater/CH₃CN (30:70).

Example 8 Synthesis of 7-Deazaadenosine Analogs Synthesis of7-(2-nitrobenzyloxy)methyl-7-deaza-2′-deoxyadenosine-5′-triphosphate

6-Chloro-7-iodo-7-deazapurine (dA.23): Compound dA.23 was synthesizedaccording to the procedure described by Ju et al. (2006, which isincorporated herein by reference). To a suspension of6-chloro-7-deazapurine (1.00 g, 6.51 mmol) in anhydrous CH₂Cl₂ (55 mL)was added N-iodosuccinimide (1.70 g, 7.56 mmol). The reaction wasprotected from light while stirring at room temperature for two hours.The reaction was then concentrated down in vacuo. The material wasre-crystallized from hot methanol to yield 6-chloro-7-iodo-7-deazapurinedA.23 (0.94 g, 52%).

9-(β-D-2′-Deoxyribofuranosyl)-6-chloro-7-iodo-7-deazapurine (dA.24):Compound dA.24 was synthesized according to the procedure described byJu et al. (2006, which is incorporated herein by reference). To asuspension of KOH (0.52 g, 8.29 mmol) and tris(3,6-dioxaheptyl)amine(0.07 mL, 0.22 mmol) in 56 mL anhydrous acetonitrile was added compounddA.23 (0.93 g, 3.32 mmol). The reaction stirred at room temperature forfive minutes and then 2-deoxy-3,5-di-O-(p-toluoyl)-α-D-ribofuranosylchloride (1.38 g, 3.55 mmol) was added over 15 minutes. The reactionstirred at room temperature for one hour then was filtered and washedwith hot acetone (50 mL). The filtrated was concentrated down in vacuoand half of the material was dissolved in 7N NH₃ in methanol solution(40 mL) and stirred at room temperature for 16 hours. The reaction wasthen concentrated down in vacuo and purified by silica gelchromatography to yield9-(β-D-2′-deoxyribofuranosyl)-6-chloro-7-iodo-7-deazapurine dA.24 (0.31g, 47%) as a white foam.

9-[β-D-3′,5′-O-Bis-(tert-butyldimethylsilyl)-2′-deoxyribofuranosyl]-6-chloro-7-iodo-7-deazapurine(dA.25): Compound dA.24 (0.30 g, 0.76 mmol) was evaporated fromanhydrous pyridine (2 mL) three times and dissolved in anhydrous DMF (5mL). tert-Butyldimethylsilyl chloride (0.34 g, 2.28 mmol) and imidazole(0.31 g, 4.55 mmol) were added and the mixture was stirred at roomtemperature for 16 hours. The reaction was concentrated in vacuo andpurified by silica gel chromatography to yield9-[β-D-3′,5′-β-bis-(tert-butyldimethylsilyl)-2′-deoxyribofuranosyl]-6-chloro-7-iodo-7-deazapurinedA.25 (0.24 g, 51%) as a white foam. ¹H NMR (400 MHz, CDCl₃): δ 8.61 (s,1 H, H-2), 7.81 (s, 1 H, H-8), 6.74 (t, 1 H, J=6.4 Hz, H-1′), 4.56 (m, 1H, H-4′), 4.01 (m, 1 H, H-3′), 3.87 (dd, 1 H, H-5′a), 3.79 (dd, 1 H,H-5′b), 2.39 (m, 2 H, H-2′a and H-2′b), 0.96 (s, 9 H, (CH₃)₃CSi), 0.91(s, 9 H, (CH₃)₃CSi), 0.18 (2s, 6 H, (CH₃)₂Si), 0.15 (s, 6 H, (CH₃)₂Si);¹³C NMR (100 MHz, CDCl₃): δ 152.50 (C), 150.80 (CH), 150.48 (C), 131.94(CH), 117.33 (C), 87.92 (CH), 84.16 (CH), 72.20 (CH), 63.01 (CH₂), 51.98(C), 42.08 (CH₂), 26.07 (CH₃), 25.77 (CH₃), 18.51 (C), 18.05 (C), −4.63(CH₃), −4.78 (CH₃), −5.25 (CH₃), −5.39 (CH₃).

9-[β-D-3′,5′-O-Bis-(tert-butyldimethylsilyl)-2′-deoxyribofuranosyl]-6-chloro-7-methoxycarbonyl-7-deazapurine(dA.26): To a solution of dA.25 (1.3 g, 2.1 mmol) in anhydrous1,4-dioxane (30 mL) and anhydrous methanol (25 mL) was addedtriethylamine (0.58 mL). After stirring for ten minutes under COatmosphere, bis(benzonitrile)dichloropalladium(II) was added. Thereaction was stirred at 50° C. for 48 hours under CO atmosphere, andthen concentrated in vacuo. The residue was purified by silica gelchromatography to give9-[β-D-3′,5′-O-bis-(tert-butyldimethylsilyl)-2′-deoxyribofuranosyl]-6-chloro-7-methoxycarbonyl-7-deazapurinedA.26 (1.15 g, 99%) as a viscous oil. ¹H NMR (400 MHz, CDCl₃): δ 8.69(s, 1 H, H-2), 8.31 (s, 1 H, H-8), 6.77 (t, 1 H, J=6.8 Hz, H-1′), 4.58(m, 1 H, H-4′), 4.06 (m, 1 H, H-3′), 3.90 (s, 3 H, CH₃O), 3.87 (dd, 1 H,H-5′a), 3.81 (dd, 1 H, H-5′b), 2.42 (m, 2 H, H-2′a and H-2′b), 0.93 (s,18 H, (CH₃)₃CSi), 0.13 (s, 6 H, (CH₃)₂Si), 0.12 (s, 6 H, (CH₃)₂Si); ¹³CNMR (100 MHz, CDCl₃): δ 162.46 (C), 153.12 (C), 152.07 (C), 151.35 (CH),133.27 (CH), 115.27 (C), 107.65 (C), 88.23 (CH), 84.52 (CH), 72.46 (CH),63.06 (CH₂), 51.55 (CH₃), 42.15 (CH₂), 25.99 (CH₃), 25.77 (CH₃), 18.45(C), 18.03 (C), −4.64 (CH₃), −4.78 (CH₃), −5.49 (CH₃), −5.55 (CH₃).

9-[β-D-3′,5′-O-Bis-(tert-butyldimethylsilyl)-2′-deoxyribofuranosyl]-6-chloro-7-hydroxymethyl-7-deazapurine(dA.27): To a solution of dA.26 (0.28 g, 0.50 mmol) in anhydrous THF (4mL) lithium borohydride (0.044 g, 2.01 mmol) was added, followed bymethanol (0.1 mL). The reaction mixture was stirred at room temperaturefor ten minutes and then heated at reflux for 45 minutes. Upon coolingdown, the reaction mixture was diluted with dichloromethane (20 ml) andquenched with water (2 mL). The organic layer was separated, washed withbrine two times (5 mL each), dried over anhydrous sodium sulfate, andconcentrated in vacuo. The residue was purified by silica gelchromatography to yield9-[β-D-3′,5′-O-bis-(tert-butyldimethylsilyl)-2′-deoxyribofuranosyl]-6-chloro-7-hydroxymethyl-7-deazapurinedA.27 (0.12 g, 45%) as a white foam. ¹H NMR (400 MHz, CDCl₃): δ 8.62 (s,1 H, H-8), 7.61 (s, 1 H, H-2), 6.75 (dd, 1 H, J=6.0 and 7.2 Hz, H-1′),4.96 (AB d, 1 H, J=11.6 Hz, 7-CH₂a), 4.91 (AB d, 1 H, J=11.6 Hz,7-CH₂b), 4.57 (m, 1 H, H-4′), 4.00 (m, 1 H, H-3′), 3.80 (m, 2 H, H-5′aand H-5′b), 2.44 (m, 1 H, H-2′a), 2.04 (m, 1 H, H-2′b), 0.91 (2 s, 18 H,(CH₃)₃CSi), 0.11 (2 s, 12 H, (CH₃)₂Si); ¹³C NMR (100 MHz, CDCl₃): δ151.84 (C), 151.37 (C), 150.98 (CH), 125.33 (CH), 115.97 (C), 115.48(C), 87.67 (CH), 83.73 (CH), 72.28 (CH), 63.07 (CH₂), 56.89 (CH₂), 41.42(CH₂), 25.98 (CH₃), 25.79 (CH₃), 18.45 (C), 18.03 (C), −4.64 (CH₃),−4.76 (CH₃), −5.35 (CH₃), −5.47 (CH₃).

9-[β-D-3′,5′-O-Bis-(tert-butyldimethylsilyl)-2′-deoxyribofuranosyl]-6-chloro-7-(2-nitrobenzyloxy)methyl-7-deazapurine(dA.28): To a solution of dA.27 (30 mg, 0.057 mmol) in CH₂Cl₂ (2 mL)were added n-Bu₄NBr (9 mg, 0.029 mmol), 2-nitrobenzyl bromide (37 mg,0.17 mmol) and 1 M NaOH solution (2 mL). The reaction mixture wasstirred vigorously at room temperature for 48 hours in the dark. Theorganic layer was separated, dried over Na₂SO₄, concentrated in vacuo,and purified by silica gel chromatography to yield9-[β-D-3′,5′-O-bis-(tert-butyldimethylsilyl)-2′-deoxyribofuranosyl]-6-chloro-7-(2-nitrobenzyloxy)methyl-7-deazapurinedA.28 (19 mg, 50%) as a viscous oil. ¹H NMR (400 MHz, CDCl₃): δ 8.63 (s,1 H, H-2), 8.06 (dd, 1 H, J=8.4 and 1.2 Hz, Ph-H), 7.84 (d, 1 H, J=7.6Hz, Ph-H), 7.64 (s, 1 H, H-8), 7.62 (m, 1 H, Ph-H), 7.43 (t, 1 H, Ph-H),6.75 (dd, 1 H, J=7.2 and 6.0 Hz, H-1′), 5.03 (s, 2 H, PhCH₂), 4.95 (ABd, 1 H, J=12.0 Hz, 7-CH₂a), 4.88 (AB d, 1 H, J=12.0 Hz, 7-CH₂b), 4.59(m, 1 H, H-4′), 4.00 (m, 1 H, H-3′), 3.80 (m, 2 H, H-5′a and H-5′b),2.48 (m, 1 H, H-2′a), 2.37 (m, 1 H, H-2′b), 0.92 (2 s, 18 H, (CH₃)₃CSi),0.11 (s, 6 H, (CH₃)₂Si), 0.10 (s, 6 H, (CH₃)₂Si).

7-(2-Nitrobenzyloxy)methyl-7-deaza-2′-deoxyadenosine (dA.29): A solutionof n-Bu₄NF (17 mg, 0.054 mmol) in THF (1 mL) was added to a solution ofdA.28 (18 mg, 0.027 mmol) in THF (1 mL) at 0° C. The reaction mixturewas gradually warmed to room temperature and stirred for two hours. Themixture was concentrated in vacuo, and then the residue was dissolved in1,4-dioxane (2 mL) followed by addition of 7N NH₃ in methanol (4 mL).The mixture was transferred to a sealed tube and stirred at 90-100° C.for 16 hours, then cooled down, concentrated in vacuo, and the residuewas purified by silica gel chromatography to yield7-(2-nitrobenzyloxy)methyl-7-deaza-2′-deoxyadenosine dA.29 (10 mg, 91%)as a white foam. ¹H NMR (400 MHz, DMSO-d₆): δ 8.08 (s, 1 H, H-2), 8.06(m, 1 H, Ph-H), 7.75 (m, 2 H, Ph-H), 7.58 (m, 1 H, Ph-H), 7.42 (s, 1 H,H-8), 6.64 (bs, 2 H, D₂O exchangeable, 6-NH₂), 6.48 (dd, 1 H, J=2.0 and6.0 Hz, H-1′), 5.25 (d, 1 H, J=4.0 Hz, D₂O exchangeable, 3′-OH), 5.08(t, 1 H, J=5.6 Hz, D₂O exchangeable, 5′-OH), 4.90 (s, 2 H, PhCH₂), 4.75(AB dd, 2 H, 7-CH₂), 4.33 (m, 1 H, H-3′), 3.81 (m, 1 H, H-4′), 3.54 (m,2 H, H-5′a and H-5′b), 2.47 (m, 1 H, H-2′a), 2.15 (m, 1 H, H-2′b).

7-(2-Nitrobenzyloxy)methyl-7-deaza-2′-deoxyadenosine-5′-triphosphate(WW5p085)

POCl₃ (2.6 μL, 0.028 mmol) was added to a solution of dA.29 (6 mg, 0.014mmol) and proton sponge (6 mg, 0.028 mmol) in trimethylphosphate (0.25mL) at minus 40° C. and stirred for four hours. A solution ofbis-tri-n-butylammonium pyrophosphate (66 mg, 0.14 mmol) andtri-n-butylamine (28 μL) in anhydrous DMF (0.28 mL) was added. After 30minutes of stirring, triethylammonium bicarbonate buffer (1 M, pH 7.5; 1mL) was added. The reaction was stirred at room temperature for one hourand then concentrated in vacuo. The residue was dissolved in water (2mL), filtered, and purified with reverse-phase HPLC using a Perkin ElmerOD-300 C₁₈ column (7 μm, 4.6×250 mm) to yield7-(2-nitrobenzyloxy)methyl-7-deaza-2′-deoxyadenosine-5′-triphosphateWW5p085. Mobile phase: A, 100 mM triethylammonium acetate (TEAA) inwater (pH 7.0); B, 100 mM TEAA in water/CH₃CN (30:70).

Synthesis of7-[1-(2-nitrophenyl)-2-methyl-propyloxy]methyl-7-deaza-2′-deoxyadenosine-5′-triphosphate

9-[β-D-3′,5′-O-Bis-(tert-butyldimethylsilyl)-2′-deoxyribofuranosyl]-6-chloro-7-chloromethyl-7-deazapurine(dA.30): To a solution of dA.27 (0.257 g, 0.485 mmol) in dichloromethane(12 mL, freshly distilled from CaH₂) were added4-N,N-dimethylaminopyridine (0.148 g, 1.213 mmol) and tosyl chloride(0.111 g, 0.583 mmol). The reaction mixture was stirred at roomtemperature for 18 hours, and then concentrated in vacuo. The residuewas purified by silica gel chromatography to yield9-[β-D-3′,5′-O-bis-(tert-butyldimethylsilyl)-2′-deoxyribofuranosyl]-6-chloro-7-chloromethyl-7-deazapurinedA.30 (0.103 g, 26%) as a viscous oil. ¹H NMR (400 MHz, CDCl₃): δ 8.64(s, 1 H, H-2), 7.72 (s, 1 H, H-8), 6.73 (t, 1 H, J=6.8 Hz, H-1′), 4.95(AB d, J=12.4 Hz, 7-CH₂a), 4.91 (AB d, J=12.0 Hz, 7-CH₂b), 4.58 (m, 1 H,H-3′), 4.00 (m, 1 H, H-4′), 3.82 (m, 2 H, H-5′a and H-5′b), 2.41 (m, 2H, H-2′a and H-2′b), 0.95 (s, 9 H, (CH₃)₃CSi), 0.93 (s, 9 H, (CH₃)₃CSi),0.12 (s, 6 H, (CH₃)₂Si), 0.11 (s, 6 H, (CH₃)₂Si); ¹³C NMR (100 MHz,CDCl₃): δ 151.78 (C), 151.56 (C), 151.26 (CH), 126.68 (CH), 112.15 (C),115.54 (C), 87.78 (CH), 83.97 (CH), 72.17 (CH), 62.98 (CH₂), 41.72(CH₂), 37.56 (CH₂), 25.99 (CH₃), 25.78 (CH₃), 18.45 (C), 18.03 (C),−4.63 (CH₃), −4.78 (CH₃), −5.35 (CH₃), −5.45 (CH₃).

9-[β-D-3′,5′-O-Bis-(tert-butyldimethylsilyl)-2′-deoxyribofuranosyl]-6-chloro-7-[1-(2-nitrophenyl)-2-methyl-propyloxy]methyl-7-deazapurine(dA.31): Compound dA.30 (54 mg, 0.1 mmol) and1-(2-nitrophenyl)-2-methyl-propanol (191 mg, 0.978 mmol) were dissolvedin anhydrous dichloromethane (10 mL). The solvent was removed in vacuoand the residue was heated in vacuo for one hour, then dissolved inethyl acetate and purified by silica gel chromatography to yield9-[β-D-3′,5′-O-bis-(tert-butyldimethylsilyl)-2′-deoxyribofuranosyl]-6-chloro-7-[1-(2-nitrophenyl)-2-methyl-propyloxy]methyl-7-deazapurinedA.31 (38 mg, 54%, 1:1 mixture of diastereomers). ¹H NMR (400 MHz,CDCl₃) for diastereomers: δ 8.60 and 8.59 (2 s, 1 H, H-2), 7.83 (m, 1 H,Ph-H), 7.79 (m, 1 H, Ph-H), 7.56 (m, 1 H, Ph-H), 7.48 and 7.47 (2 s, 1H, H-8), 7.38 (m, 1 H, Ph-H), 6.70 (m, 1 H, H-1′), 4.81 (m, 1 H, Ph-CH),4.70 (m, 1 H, 7-CH₂a), 4.58 (m, 2 H, 7-CH₂b and H-3′), 3.99 (m, 1 H,H-4′), 3.78 (m, 2 H, H-5′a and H-5′b), 2.48 (m, 1 H, H-2′a), 2.35 (m, 1H, H-2′b), 1.96 (m, 1 H, CH), 0.98 and 0.96 (2 d, 3 H, CH₃), 0.93 (2 s,9 H, (CH₃)₃CSi), 0.89 (2 s, 9 H, (CH₃)₃CSi), 0.82 and 0.78 (2 d, 3 H,CH₃), 0.12 (2 s, 6 H, (CH₃)₂Si), 0.08 and 0.07 (2 s, 3 H, (CH₃)₂Si),0.06 and 0.05 (2 s, 3 H, (CH₃)₂Si); ¹³C NMR (100 MHz, CDCl₃) fordiastereomers: δ 152.60 and 152.47 (C), 150.84 (CH), 150.28 and 150.21(C), 149.56 and 149.47 (C), 148.01 (C), 137.22 and 137.08 (C), 132.70and 132.68 (CH), 129.15 and 129.13 (CH), 127.97 (CH), 126.65 and 126.29(CH), 123.85 and 123.79 (CH), 112.36 and 112.07 (C), 87.63 and 87.59(CH), 83.71 and 83.68 (CH), 81.92 and 81.08 (CH), 72.40 and 72.28 (CH),63.50 (CH₂), 63.15 and 63.03 (CH₂), 41.07 and 41.00 (CH₂), 35.08 and35.05 (CH), 19.20 and 19.11 (CH₃), 18.42 and 18.40 (C), 18.18 and 18.05(CH₃), −4.67 and −4.76 (CH₃Si), −4.78 (CH₃Si), −5.35 (CH₃Si), −5.47 and−5.51 (CH₃Si).

7-[1-(2-Nitrophenyl)-2-methyl-propyloxy]methyl-7-deaza-2′-deoxyadenosine(dA.32): A solution of n-Bu₄NF (44 mg, 0.140 mmol) in THF (2 mL) wasadded to a solution of dA.31 (38 mg, 0.053 mmol) in THF (2 mL) at 0° C.The reaction was gradually warmed to room temperature and stirred fortwo hours. The mixture was concentrated in vacuo, dissolved in1,4-dioxane (4 mL), followed by addition of 7N NH₃ in methanol solution(8 mL). The mixture was transferred to a sealed tube and stirred at90-100° C. for 24 hours, then cooled down, concentrated in vacuo, andthe residue was purified by silica gel chromatography to yield7-[1-(2-nitrophenyl)-2-methyl-propyloxy]methyl-7-deaza-2′-deoxyadenosinedA.32 (19 mg, 76%, 1:1 mixture of diastereomers) as a viscous oil. ¹HNMR (400 MHz, DMSO-d₆) for diastereomers: δ 8.06 and 8.04 (2 s, 1 H,H-2), 7.90 (m, 1 H, Ph-H), 7.67 (m, 2 H, Ph-H), 7.56 (m, 2 H, Ph-H),7.19 and 7.16 (2 s, 1 H, H-8), 6.63 (bs, 2 H, D₂O exchangeable, 6-NH₂),6.39 (m, 1 H, H-1′), 5.23 (m, 1 H, D₂O exchangeable, 3′-OH), 5.00 (m, 1H, D₂O exchangeable, 5′-OH), 4.72 (2 d, 1 H, Ph-CH), 4.45 (s, 2 H,7-CH₂), 4.30 (m, 1 H, H-3′), 3.77 (m, 1 H, H-4′), 3.49 (m, 2 H, H-5′aand H-5′b), 2.40 (m, 1 H, H-2′a), 2.12 (m, 1 H, H-2′b), 1.94 (m, 1 H,CH), 0.87 (m, 3 H, CH₃), 0.74 (m, 3 H, CH₃); ¹³C NMR (100 MHz, CD₃OD)for diastereomers: δ 157.76 (C), 151.08 (CH), 149.92 and 149.57 (C),148.01 (C), 135.99 and 135.92 (C), 132.51 and 132.41 (CH), 128.89 (CH),128.20 and 128.15 (CH), 123.49 and 123.43 (CH), 122.32 and 121.97 (CH),111.86 (C), 103.02 (C), 87.65 and 87.59 (CH), 85.25 and 85.00 (CH),80.29 and 79.60 (CH), 71.73 (CH), 63.97 and 69.92 (CH₂), 63.49 and 62.41(CH₂), 39.95 and 39.77 (CH₂), 34.55 and 34.51 (CH), 18.09 (CH₃), 17.16(CH₃).

7-[1-(2-Nitrophenyl)-2-methyl-propyloxy]methyl-7-deaza-2′-deoxyadenosine-5′-triphosphate(WW5p098 ds1 & ds2): POCl₃ (8 μL, 0.083 mmol) was added to a solution ofcompound dA.32 (19 mg, 0.041 mmol) in trimethylphosphate (0.4 mL), andthe reaction was stirred at minus 40° C. under a nitrogen atmosphere fortwo hours. Additional POCl₃ (8 μL, 0.083 mmol) was added, and thereaction was stirred at 0° C. for additional three hours. A solution ofbis-tri-n-butylammonium pyrophosphate (97 mg, 0.2 mmol) andtri-n-butylamine (40 μL) in anhydrous DMF (0.4 mL) was added. After 30minutes of stirring, triethylammonium bicarbonate buffer (1 M, pH 7.5;10 mL) was added. The reaction was stirred for one hour at roomtemperature and then concentrated in vacuo. The residue was dissolved inwater (5 mL), filtered, and purified by anion exchange chromatographyusing a Q Sepharose FF column (2.5×10 cm) with a linear gradient of 25%acetonitrile/75% 0.1M triethylammonium bicarbonate (TEAB) to 25%acetonitrile/75% 1.5 M TEAB over 240 min at 4.5 ml/min. The fractionscontaining triphosphate were combined and lyophilized to yield7-[1-(2-nitrophenyl)-2-methyl-propyloxy]methyl-7-deaza-2′-deoxyadenosine-5′-triphosphateWW5p098 as mixture of two diastereomers, which were separated by reversephase HPLC on a Perkin Elmer Aquapore OD-300 column (7 μm, 250×4.6 mm)to yield the single diastereomer WW5p098 ds1 (fast eluting) and WW5p098ds2 (slow eluting). Mobile phase: A, 100 mM triethylammonium acetate(TEAA) in water; B, 100 mM TEAA in water/CH₃CN (30:70).

Synthesis of 6-FAM labeled7-{(R)-1-[4-(3-amino-1-propynyl)-2-nitrophenyl]-2-methyl-propyloxy}methyl-7-deaza-2′-deoxyadenosine-5′-triphosphate

9-[β-D-3′,5′-O-Bis-(tert-butyldimethylsilyl)-2′-deoxyribofuranosyl]-6-chloro-7-[(R)-1-(4-iodo-2-nitrophenyl)-2-methyl-propyloxy]methyl-7-deazapurine(dA.33): Compound dA.30 (80 mg, 0.147 mmol) and enantio-pure(R)-1-(4-iodo-2-nitrophenyl)-2-methyl-propanol (518 mg, 1.163 mmol) weredissolved in anhydrous dichloromethane (10 mL). The solvent was removedin vacuo, and the residue was heated in vacuo for one hour, thendissolved in ethyl acetate and purified by silica gel chromatography toyield9-[β-D-3′,5′-O-bis-(tert-butyldimethylsilyl)-2′-deoxyribofuranosyl]-6-chloro-7-[(R)-1-(4-iodo-2-nitrophenyl)-2-methyl-propyloxy]methyl-7-deazapurinedA.33 (57 mg, 47%). ¹H NMR (400 MHz, CDCl₃): δ 8.60 (s, 1 H, H-2), 8.12(d, J=2.0 Hz, 1 H, Ph-H), 7.87 (dd, J=8.4 and 1.6 Hz, 1 H, Ph-H), 7.47(d, J=8.0 Hz, 1 H, Ph-H), 7.47 (s, 1 H, H-8), 6.71 (dd, J=7.6 and 6.0Hz, 1 H, H-1′), 4.76 (d, J=6.4 Hz, 1 H, Ph-CH), 4.70 (AD d, J=11.6 Hz, 1H, 7-CH₂a), 4.58 (m, 2 H, 7-CH₂b and H-3′), 4.00 (m, 1 H, H-4′), 3.79(m, 2 H, H-5′a and H-5′b), 2.45 (m, 1 H, H-2′a), 2.36 (m, 1 H, H-2′b),1.93 (sep, J=6.8 Hz, 1 H, CHCH(CH₃)₂), 0.98 (d, J=6.4 Hz, 3 H, CH₃),0.93 (s, 9 H, (CH₃)₃CSi), 0.91 (s, 9 H, (CH₃)₃CSi), 0.82 (d, J=6.8 Hz, 3H, CH₃), 0.126 (s, 6 H, (CH₃)₂Si), 0.123 (s, 3 H, (CH₃)₂Si), 0.09 (s, 3H, (CH₃)₂Si), 0.06 (s, 3 H, (CH₃)₂Si); ¹³C NMR (100 MHz, CDCl₃): δ151.81 (C), 151.76 (C), 150.93 (CH), 149.79 (C), 141.63 (CH), 137.04(C), 132.34 (CH), 130.85 (CH), 126.41 (CH), 116.17 (C), 112.06 (C),91.57 (C), 87.65 (CH), 83.77 (CH), 80.68 (CH), 72.41 (CH), 63.58 (CH₂),63.15 (CH₂), 41.07 (CH₂), 34.95 (CH), 25.96 (C(CH₃)₃), 25.80 (C(CH₃)₃),19.13 (CH₃), 18.42 (C), 18.05 (CH₃), −4.63 (CH₃), −4.78 (CH₃), −5.35(CH₃), −5.45 (CH₃).

7-[(R)-1-(4-Iodo-2-nitrophenyl)-2-methyl-propyloxy]methyl-7-deaza-2′-deoxyadenosine(dA.34): A solution of n-Bu₄NF (58 mg, 0.182 mmol) in THF (2 mL) wasadded to a solution of dA.33 (57 mg, 0.069 mmol) in THF (2 mL) at 0° C.The reaction was gradually warmed to room temperature and stirred fortwo hours. The mixture was concentrated in vacuo, dissolved in1,4-dioxane (5 mL), followed by addition of 7N NH₃ in methanol solution(16 mL). The mixture was transferred to a sealed tube and stirred at90-100° C. for 24 hours, then cooled down, concentrated in vacuo, andthe residue was purified by silica gel chromatography to yield7-[(R)-1-(4-iodo-2-nitrophenyl)-2-methyl-propyloxy]methyl-7-deaza-2′-deoxyadenosinedA.34 (33 mg, 82%). ¹H NMR (400 MHz, DMSO-d₆): δ 8.22 (d, J=1.6 Hz, 1 H,Ph-H), 8.04 (s, 1 H, H-2), 8.00 (dd, J=8.4 and 1.6 Hz, 1 H, Ph-H), 7.39(d, J=8.4 Hz, 2 H, Ph-H), 7.19 (s, 1 H, H-8), 6.60 (bs, 2 H, D₂Oexchangeable, 6-NH₂), 6.40 (dd, J=8.4 and 6.0 Hz, 1 H, H-1′), 5.24 (d,J=4.0 Hz, 1 H, D₂O exchangeable, 3′-OH), 5.00 (d, J=5.2 Hz, 1 H, D₂Oexchangeable, 5′-OH), 4.64 (d, J=6.0 Hz, 1 H, Ph-CH), 4.45 (AB dd, 2H,7-CH₂), 4.29 (m, 1 H, H-3′), 3.78 (m, 1 H, H-4′), 3.47 (m, 2 H, H-5′aand H-5′b), 2.40 (m, 1 H, H-2′a), 2.11 (m, 1 H, H-2′b), 1.92 (m, 1 H,CH), 0.87 (d, J=6.4 Hz, 3 H, CH₃), 0.76 (d, J=6.8 Hz, 3 H, CH₃); ¹³C NMR(100 MHz, CD₃OD): δ 164.68 (C), 151.12 (CH), 150.12 (C), 149.84 (C),141.38 (CH), 136.09 (C), 131.94 (CH), 130.66 (CH), 122.15 (CH), 111.79(C), 103.09 (C), 91.16 (C), 87.63 (CH), 85.12 (CH), 80.31 (CH), 71.80(CH), 63.36 (CH₂), 62.53 (CH₂), 39.76 (CH₂), 34.41 (CH), 18.00 (CH₃),17.20 (CH₃).

7-{(R)-1-[4-(3-Trifluoroacetamido-1-propynyl)-2-nitrophenyl]-2-methyl-propyloxy}methyl-7-deaza-2′-deoxyadenosine(dA.35): A solution of compound dA.34 (33 mg, 0.056 mmol),N-propargyltrifluoroacetylamide (25 mg, 0.168 mmol),tetrakis(triphenylphosphine)-palladium(0) (7 mg, 0.0065 mmol), CuI (2mg, 0.0112 mmol), and Et₃N (16 μL, 0.050 mmol) in anhydrous DMF (3 mL)was stirred at room temperature for four hours. The mixture wasconcentrated in vacuo and purified by silica gel column chromatographyto yield7-{(R)-1-[4-(3-trifluoroacetamido-1-propynyl)-2-nitrophenyl]-2-methyl-propyloxy}methyl-7-deaza-2′-deoxyadenosinedA.35 (34 mg, 99%) as a waxy solid. ¹H NMR (400 MHz, DMSO-d₆): δ 10.11(br t, 1 H, D₂O exchangeable, NHTFA), 8.12 (br s, 1 H, H-2), 7.94 (d,J=1.6 Hz, 1 H, Ph-H), 7.71 (AB dd, J=8.0 and 1.6 Hz, 1 H, Ph-H), 7.62(AB d, J=8.4 Hz, 2 H, Ph-H), 7.28 (s, 1 H, H-8), 6.95 (bs, 2 H, D₂Oexchangeable, 6-NH₂), 6.42 (dd, J=8.0 and 6.0 Hz, 1 H, H-1′), 5.25 (brs, 1 H, D₂O exchangeable, 3′-OH), 4.98 (br s, 1 H, D₂O exchangeable,5′-OH), 4.60 (d, J=6.0 Hz, 1 H, Ph-CH), 4.51 (AB dd, J=12.8 Hz, 1 H,7-CH₂a), 4.45 (AB dd, J=12.4 Hz, 1 H, 7-CH₂b), 4.30 (m, 3 H, CH ₂NH andH-3′), 3.78 (m, 1 H, H-4′), 3.47 (m, 2 H, H-5′a and H-5′b), 2.39 (m, 1H, H-2′a), 2.14 (m, 1 H, H-2′b), 1.95 (m, 1 H, CH), 0.88 (d, J=6.8 Hz, 3H, CH₃), 0.76 (d, J=6.8 Hz, 3 H, CH₃); ¹³C NMR (100 MHz, CD₃OD): δ157.62 (C), 151.15 (CH), 150.05 (C), 149.47 (C), 136.64 (C), 135.03(CH), 129.34 (CH), 126.31 (CH), 122.68 (C), 122.14 (CH), 115.02 (C),111.84 (C), 87.59 (CH), 85.83 (C), 85.01 (CH), 80.28 (CH), 80.13 (C),81.42 (CH), 71.74 (CH), 64.28 (CH₂), 62.49 (CH₂), 39.73 (CH₂), 34.50(CH), 29.07 (CH₂), 18.03 (CH₃), 17.18 (CH₃).

7-{(R)-1-[4-(3-Amino-1-propynyl)-2-nitrophenyl]-2-methyl-propyloxy}methyl-7-deaza-2′-deoxyadenosine-5′-triphosphate(dA.36): POCl₃ (8 μL, 0.089 mmol) was added to a solution of compounddA.35 (27 mg, 0.045 mmol) and proton sponge (19 mg, 0.089 mmol) intrimethylphosphate (0.4 mL) and the reaction was stirred at 0° C. undera nitrogen atmosphere for two hours. A solution ofbis-tri-n-butylammonium pyrophosphate (285 mg, 0.6 mmol) andtri-n-butylamine (120 μL) in anhydrous DMF (1.2 mL) was added. After 30minutes of stirring, triethylammonium bicarbonate buffer (1 M, pH 7.5;10 mL) was added. The reaction was stirred for one hour at roomtemperature and then concentrated in vacuo. The residue was dissolved inwater (10 mL), filtered, and purified by anion exchange chromatographyusing a Q Sepharose FF column (2.5×10 cm) with a linear gradient of 25%acetonitrile/75% 0.1 M triethylammonium bicarbonate (TEAB) to 25%acetonitrile/75% 1.5 M TEAB over 240 min at 4.5 ml/min. The fractionscontaining triphosphate were combined and lyophilized to dryness. Theresidue was dissolved in water (5 mL) and treated with concentratedammonium hydroxide (2 mL, 27%) at room temperature for one hour to yield7-{(R)-1-[4-(3-amino-1-propynyl)-2-nitrophenyl]-2-methyl-propyloxy}methyl-7-deaza-2′-deoxyadenosine-5′-triphosphatedA.36, which was purified by reverse phase HPLC on a Perkin ElmerAquapore OD-300 column (7 μm, 250×4.6 mm). Mobile phase: A, 100 mMtriethylammonium acetate (TEAA) in water; B, 100 mM TEAA in water/CH₃CN(30:70).

6-FAM labeled7-{(R)-1-[4-(3-amino-1-propynyl)-2-nitrophenyl]-2-methyl-propyloxy}methyl-7-deaza-2′-deoxyadenosine-5′-triphosphate(WW6p028): A solution of 6-FAM-SE (4 mg, 8.4 μmol) in anhydrous DMSO (80μL) was added to a solution of triphosphate dA.36 (2.6 μmol) inNa₂CO₃/NaHCO₃ buffer (0.1 M, pH 9.2, 1.6 mL). The mixture was left atroom temperature for one hour. The dye labeled triphosphate was firstpurified by anion exchange HPLC using a PerkinElmer AX-300 column (7 μm,250×4.6 mm). Mobile phase: A, 25% CH₃CN/75% 0.1 M TEAB; B, 25% CH₃CN/75%1.5 M TEAB. The product was further purified by reverse-phase HPLC usinga Perkin Elmer OD-300 column (7 μm, 4.6×250 mm) to yield 6-FAM labeledtriphosphate WW6p028. Mobile phase: A, 100 mM triethylammonium acetate(TEAA) in water (pH 7.0); B, 100 mM TEAA in water/CH₃CN (30:70).

Synthesis of7-[1-(2,6-dinitrophenyl)-2-methyl-propyloxy]methyl-7-deaza-2′-deoxyadenosine-5′-triphosphate

9-[β-D-3′-O-(tert-Butyldimethylsilyl)-2′-deoxyribofuranosyl]-6-chloro-7-[1-(2,6-dinitrophenyl)-2-methyl-propyloxy]methyl-7-deazapurine(dA.37a) and9-[β-D-2′-deoxyribofuranosyl]-6-chloro-7-[1-(2,6-dinitrophenyl)-2-methyl-propyloxy]methyl-7-deazapurine(dA.37b): Compound dA.30 (109 mg, 0.201 mmol) and1-(2,6-dinitrophenyl)-2-methyl-propanol (448 mg, 1.863 mmol) weredissolved in anhydrous dichloromethane (10 mL). The solvent was removedin vacuo, and the residue was heated at 108° C. in vacuo for 30 minutes,then dissolved in ethyl acetate and purified by silica gelchromatography to yield9-[β-D-3′-O-(tert-butyldimethylsilyl)-2′-deoxyribofuranosyl]-6-chloro-7-[1-(2,6-dinitrophenyl)-2-methyl-propyloxy]methyl-7-deazapurinedA.37a (42 mg, 33%, 1:1 mixture of diastereomers) and9-[β-D-2′-deoxyribofuranosyl]-6-chloro-7-[1-(2,6-dinitrophenyl)-2-methyl-propyloxy]methyl-7-deazapurinedA.37b (13 mg, 11%, 1:1 mixture of diastereomers). ¹H NMR (400 MHz,CDCl₃) for dA.37a (1:1 mixture of diastereomers): δ 8.59 (s, 1 H, H-2),7.78 (m, 2 H, Ph-H), 7.63 (m, 1 H, Ph-H), 7.38 (s, 1 H, H-8), 6.39 (m, 1H, H-1′), 5.01 (2 br s, 1 H, 5′-OH), 4.72 (m, 4 H, Ph-CH, 7-CH₂, andH-3′), 4.11 (m, 1 H, H-4′), 3.91 (AB d, 1 H, H-5′a), 3.85 (m, 1 H,H-5′b), 2.96 (m, 1 H, CH(CH₃)₂), 2.40 (m, 2 H, H-2′), 1.08 (m, 3 H,CH₃), 0.91 (s, 9 H, (CH₃)₃CSi), 0.78 (2 d, J=6.8 Hz, 3 H, CH₃), 0.13 (s,6 H, (CH₃)₂Si); ¹³C NMR (100 MHz, CDCl₃) for dA.37a (1:1 mixture ofdiastereomers): δ 152.60 and 152.47 (C), 150.92 and 150.66 (C), 150.47and 150.28 (C), 151.84 (CH), 150.28 and 150.21 (C), 129.72 and 129.69(CH), 129.18 (CH), 128.77 (C), 128.63 and 128.54 (CH), 126.69 (CH),117.57 and 117.29 (C), 110.84 and 110.59 (C), 89.39 and 89.18 (CH),88.70 and 88.80 (CH), 82.00 and 81.84 (CH), 73.47 and 73.35 (CH), 64.56and 64.34 (CH₂), 63.10 and 63.05 (CH₂), 41.12 (CH₂), 34.86 (CH), 25.81((CH₃)₃Si), 19.13 (CH₃), 18.36 (CH₃), 18.08 (C), −4.67 (CH₃Si), −4.76(CH₃Si).

¹H NMR (400 MHz, CD₃OD) for dA.37b (1:1 mixture of diastereomers): δ8.57 and 8.56 (2 s, 1 H, H-2), 7.96 (m, 2 H, Ph-H), 7.45 (m, 2 H, Ph-Hand H-8), 6.39 (m, 1 H, H-1′), 4.78 (m, 2 H, Ph-CH, 7-CH₂a), 4.56 (m, 2H, 7-CH₂b and H-3′), 4.02 (m, 1 H, H-4′), 3.78 (m, 2 H, H-5′), 2.62 (m,1 H, CH(CH₃)₂), 2.44 (m, 1 H, H-2′a), 2.30 (m, 1 H, H-2′b), 1.02 and0.95 (2 d, J=6.4 Hz, 3 H, CH₃), 0.71 and 0.69 (2 d, J=7.2 Hz, 3 H, CH₃).

7-[1-(2,6-Dinitrophenyl)-2-methyl-propyloxy]methyl-7-deaza-2′-deoxyadenosine(dA.38): A solution of n-Bu₄NF (44 mg, 0.140 mmol) in THF (2 mL) wasadded to a solution of dA.37a (42 mg, 0.066 mmol) in THF (5 mL) at 0° C.The reaction was gradually warmed to room temperature and stirred fortwo hours. The mixture was concentrated in vacuo, and a solution ofdA.37b (12 mg, 0.022 mmol) in 1,4-dioxane (4 mL) was added, followed by7N NH₃ in methanol solution (18 mL). The mixture was transferred to asealed tube and stirred at 90-100° C. for 36 hours, then cooled down,concentrated in vacuo, and the residue was purified by silica gelchromatography to yield7-[1-(2,6-dinitrophenyl)-2-methyl-propyloxy]methyl-7-deaza-2′-deoxyadenosinedA.38 (38 mg, 86%, 1:1 mixture of diastereomers) as a viscous oil. ¹HNMR (400 MHz, DMSO-d₆) for diastereomers: δ 8.17 (m, 1 H, Ph-H), 8.07and 8.06 (2 s, 1 H, H-2), 7.85 (m, 1 H, Ph-H), 7.69 (m, 1 H, Ph-H), 7.20and 7.18 (2 s, 1 H, H-8), 6.57 (bs, 2 H, D₂O exchangeable, 6-NH₂), 6.46(m, 1 H, H-1′), 5.26 (d, J=3.6 Hz, 1 H, D₂O exchangeable, 3′-OH), 5.01(m, 1 H, D₂O exchangeable, 5′-OH), 4.60 (m, 2 H, Ph-CH and 7-CH₂a), 4.29(m, 1 H, 7-CH₂b), 4.13 (m, 1 H, H-3′), 3.80 (m, 1 H, H-4′), 3.51 (m, 2H, H-5′a and H-5′b), 2.49 (m, 1 H, CH(CH₃)₃), 2.16 (m, 1 H, H-2′a andH-2′b), 0.91 (m, 3 H, CH₃), 0.65 (m, 3 H, CH₃); ¹³C NMR (100 MHz, CD₃OD)for diastereomers: δ 157.73 (C), 151.33 and 151.18 (CH), 150.39 (C),150.22 (C), 130.45 and 130.49 (CH), 127.25 and 127.13 (C), 126.90 (CH),128.20 and 128.15 (CH), 123.45 and 123.32 (CH), 110.32 and 110.23 (CH),103.03 and 102.75 (C), 87.74 and 87.58 (CH), 85.43 and 84.73 (CH), 79.94and 79.37 (CH), 71.88 and 71.64 (CH), 64.08 and 63.71 (CH₂), 62.67 and62.32 (CH₂), 40.95 and 39.82 (CH₂), 34.24 and 34.16 (CH), 19.63 (CH₃),17.49 (CH₃). ToF-MS (ESI): For the molecular ion C₂₂H₂₇N₆O₈ [M+H]⁺, thecalculated mass was 503.1890, and the observed mass was 503.2029.

7-[1-(2,6-Dinitrophenyl)-2-methyl-propyloxy]methyl-7-deaza-2′-deoxyadenosine-5′-triphosphate(WW6p057 ds1 & ds2): POCl₃ (11 μL, 0.12 mmol) was added to a solution ofcompound dA.38 (30 mg, 0.06 mmol) in trimethylphosphate (0.4 mL) and thereaction was stirred at 0° C. under a nitrogen atmosphere for fourhours. A solution of bis-tri-n-butylammonium pyrophosphate (285 mg, 0.6mmol) and tri-n-butylamine (120 μL) in anhydrous DMF (1.2 mL) was added.After 30 minutes of stirring, triethylammonium bicarbonate buffer (1 M,pH 7.5; 10 mL) was added. The reaction was stirred for one hour at roomtemperature and then concentrated in vacuo. The residue was dissolved inwater (10 mL), filtered, and purified by anion exchange chromatographyusing a Q Sepharose FF column (2.5×20 cm) with a linear gradient of 25%acetonitrile/75% 0.1 M triethylammonium bicarbonate (TEAB) to 25%acetonitrile/75% 1.5 M TEAB over 240 min at 4.5 ml/min. The fractionscontaining triphosphate were combined and lyophilized to yield7-[1-(2,6-dinitrophenyl)-2-methyl-propyloxy]methyl-7-deaza-2′-deoxyadenosine-5′-triphosphateWW6p057 as mixture of two diastereomers which were separated by reversephase HPLC on a PerkinElmer Aquapore OD-300 column (7 μm, 250×4.6 mm) toyield the single diastereomer WW6p057 ds1 (fast eluting) and WW6p057 ds2(slow eluting). Mobile phase: A, 100 mM triethylammonium acetate (TEAA)in water; B, 100 mM TEAA in water/CH₃CN (30:70).

Synthesis of7-[1-(4-methoxy-2-nitrophenyl)-2-methyl-propyloxy]methyl-7-deaza-2′-deoxyadenosine-5′-triphosphate

9-[β-D-3′,5′-O-Bis-(tert-butyldimethylsilyl)-2′-deoxyribofuranosyl]-6-chloro-7-[1-(4-methoxy-2-nitrophenyl)-2-methyl-propyloxy]methyl-7-deazapurine(dA.39a) and9-[β-D-3′-O-(tert-butyldimethylsilyl)-2′-deoxyribofuranosyl]-6-chloro-7-[1-(4-methoxy-2-nitrophenyl)-2-methyl-propyloxy]methyl-7-deazapurine(dA.39b): Compound dA.30 (103 mg, 0.19 mmol) and1-(4-methoxy-2-nitrophenyl)-2-methyl-propanol (428 mg, 1.90 mmol) weredissolved in anhydrous dichloromethane (3 mL). The solvent was removedin vacuo, and the residue was heated at 108° C. in vacuo for 30 minutes,then dissolved in ethyl acetate and purified by silica gelchromatography to9-[β-D-3′,5′-O-bis-(tert-butyldimethylsilyl)-2′-deoxyribofuranosyl]-6-chloro-7-[1-(4-methoxy-2-nitrophenyl)-2-methyl-propyloxy]methyl-7-deazapurinedA.39a (46 mg, 33%, 1:1 mixture of diastereomers) and9-[β-D-3′-O-(tert-butyldimethylsilyl)-2′-deoxyribofuranosyl]-6-chloro-7-[1-(4-methoxy-2-nitrophenyl)-2-methyl-propyloxy]methyl-7-deazapurinedA.39b (25 mg, 21%, 1:1 mixture of diastereomers). ¹H NMR (400 MHz,CDCl₃) for dA.39a (1:1 mixture of diastereomers): δ 8.60 and 8.59 (2 s,1 H, H-2), 7.64 and 7.62 (2 d, J=6.4 Hz, 1 H, Ph-H), 7.47 and 7.45 (2 s,1 H, H-8), 7.32 (m, 1 H, Ph-H), 7.12 (m, 1 H, Ph-H), 6.71 (m, 1 H,H-1′), 4.62 (m, 4 H, Ph-CH, 7-CH₂, and H-3′), 3.99 (m, 1 H, H-4′), 3.87and 3.86 (2 s, 3 H, MeO), 3.70 (AB d, 1 H, H-5′a and H-5′b), 2.50 (m, 1H, H-2′a), 2.35 (m, 1 H, H-2′a), 1.93 (m, 1 H, CH(CH₃)₂), 1.00 and 0.97(2 d, J=6.8 Hz, 3 H, CH₃), 0.93 and 0.92 (2 s, 9 H, (CH₃)₃CSi), 0.91 and0.89 (2 s, 9 H, (CH₃)₃CSi), 0.80 and 0.76 (2 d, J=6.8 Hz, 3 H, CH₃),0.12 and 0.10 (2 s, 6 H, (CH₃)₂Si), 0.08, 0.07, 0.06 and 0.05 (4 s, 6 H,(CH₃)₂Si); ¹³C NMR (100 MHz, CDCl₃) for dA.39a (1:1 mixture ofdiastereomers): δ 158.82 (C), 151.82 and 151.46 (C), 151.32 and 151.16(C), 150.85 (CH), 150.27 and 150.08 (C), 130.16 (CH), 128.95 and 129.80(C), 126.56 and 126.22 (CH), 119.60 (CH), 112.49 and 122.22 (C), 108.21and 108.14 (CH), 87.64 and 87.58 (CH), 83.69 (CH), 80.68 and 79.98 (CH),72.42 and 72.27 (CH), 63.26 and 63.17 (CH₂), 63.03 and 62.80 (CH₂),55.80 (CH₃), 41.04 (CH₂), 35.08 (CH), 25.95 ((CH₃)₃Si), 25.80((CH₃)₃Si), 25.66 ((CH₃)₃Si), 19.12 and 19.05 (CH₃), 18.42 (CH₃), 18.05(C), −3.75 (CH₃Si), −4.66 and −4.76 (CH₃Si), −5.36 (CH₃Si), −5.46 and−5.50 (CH₃Si).

¹H NMR (400 MHz, CDCl₃) for dA.39b (1:1 mixture of diastereomers): δ8.59 and 8.57 (2 s, 1 H, H-2), 7.62 and 7.60 (2 d, J=6.4 Hz, 1 H, Ph-H),7.33 and 7.32 (2 d, J=2.4 Hz, 1 H, Ph-H), 7.30 and 7.29 (2 s, 1 H, H-8),7.14 and 7.10 (2 dd, J=8.8, 2.4 Hz, 1 H, Ph-H), 6.28 (m, 1 H, H-1′),5.06 (br t, 1 H, 5′-OH), 4.64 (m, 4 H, Ph-CH, 7-CH₂, and H-3′), 4.11 (m,1 H, H-4′), 3.94 (AB d, J=10.8 Hz, 1 H, H-5′a), 3.87 and 3.85 (2 s, 3 H,MeO), 3.76 (m, 1 H, H-5′b), 2.93 (m, 1 H, H-2′a), 2.25 (m, 1 H, H-2′b),1.96 (sep, J=6.4 Hz, 1 H, CH(CH₃)₂), 0.95 (m, 3 H, CH₃), 0.94 (s, 9 H,(CH₃)₃CSi), 0.80 (m, 3 H, CH₃), 0.13 (s, 6 H, (CH₃)₂Si).

7-[1-(4-methoxy-2-nitrophenyl)-2-methyl-propyloxy]methyl-7-deaza-2′-deoxyadenosine(dA.40): A solution of n-Bu₄NF (68 mg, 0.217 mmol) in THF (2 mL) wasadded to a solution of dA.39a (46 mg, 0.063 mmol) and dA.39b (25 mg,0.040 mmol) in THF (8 mL) at 0° C. The reaction was gradually warmed toroom temperature and stirred for 30 minutes. The mixture wasconcentrated in vacuo, dissolved in 1,4-dioxane (8 mL), followed byaddition of 7N NH₃ in methanol (24 mL). The mixture was transferred to asealed tube and stirred at 90-100° C. for 16 hours, then cooled down,concentrated in vacuo, and the residue was purified by silica gelchromatography to yield7-[1-(4-methoxy-2-nitrophenyl)-2-methyl-propyloxy]methyl-7-deaza-2′-deoxyadenosinedA.40 (38 mg, 61%, 1:1 mixture of diastereomers) as a viscous oil. ¹HNMR (400 MHz, DMSO-d₆) for diastereomers: δ 8.06 and 8.05 (2 s, 1 H,H-2), 7.57 and 7.54 (2 d, J=8.8 Hz, 1 H, Ph-H), 7.47 and 7.44 (2 d,J=2.6 Hz, 1 H, Ph-H), 7.33 and 7.27 (2 dd, J=8.8, 2.6 Hz, 1 H, Ph-H),7.18 and 7.15 (2 s, 1 H, H-8), 6.63 (bs, 2 H, D₂O exchangeable, 6-NH₂),6.43 (m, 1 H, H-1′), 5.24 (m, 1 H, D₂O exchangeable, 3′-OH), 5.03 (m, 1H, D₂O exchangeable, 5′-OH), 4.55 (m, 2 H, Ph-CH, 7-CH₂a), 4.30 (m, 2 H,7-CH₂b and H-3′), 3.86 and 3.84 (2 s, 3 H, MeO), 3.78 (m, 1 H, H-4′),3.48 (m, 2 H, H-5′), 2.45 (m, 1 H, H-2′a), 2.12 (m, 1 H, H-2′b), 1.93(m, 1 H, CH(CH₃)₂), 0.88 (m, 3 H, CH₃), 0.74 and 0.71 (2 d, J=6.8 Hz, 3H, CH₃); ¹³C NMR (100 MHz, CD₃OD) for diastereomers: δ 158.46 and 158.40(C), 158.40 (C), 150.28 and 150.25 (CH), 149.97 and 149.78 (C), 149.28(C), 129.19 and 129.16 (CH), 126.69 and 126.56 (C), 121.36 and 121.02(CH), 118.11 and 117.99 (CH), 111.20 and 110.95 (C), 107.27 and 107.20(CH), 102.40 and 102.36 (C), 86.87 and 86.83 (CH), 84.42 and 84.25 (CH),79.47 and 78.73 (CH), 70.97 (CH), 63.00 and 62.46 (CH₂), 61.73 and 61.64(CH₂), 54.30 (CH₃), 39.16 and 38.99 (CH₂), 33.71 and 33.68 (CH), 17.29(CH₃), 16.71 and 16.66 (CH₃). ToF-MS (ESI): For the molecular ionC₂₃H₃₀N₅O₇ [M+H]⁺, the calculated mass was 488.2145, and the observedmass was 488.2466.

7-[1-(4-Methoxy-2-nitrophenyl)-2-methyl-propyloxy]methyl-7-deaza-2′-deoxyadenosine-5′-triphosphate(WW6p087 ds1 & ds2)

POCl₃ (11 μL, 0.12 mmol) was added to a solution of compound dA.40 (28mg, 0.06 mmol) in trimethylphosphate (0.35 mL), and the reaction wasstirred at 0° C. under a nitrogen atmosphere for two hours. A solutionof bis-tri-n-butylammonium pyrophosphate (237 mg, 0.5 mmol) andtri-n-butylamine (100 μL) in anhydrous DMF (1.0 mL) was added. After 10minutes of stirring, triethylammonium bicarbonate buffer (1 M, pH 7.5;10 mL) was added. The reaction was stirred for one hour at roomtemperature and then concentrated in vacuo. The residue was dissolved inwater (10 mL), filtered, and purified by anion exchange chromatographyusing a Q Sepharose FF column (2.5×20 cm) with a linear gradient of 25%acetonitrile/75% 0.1 M triethylammonium bicarbonate (TEAB) to 25%acetonitrile/75% 1.5 M TEAB over 240 min at 4.5 ml/min. The fractionscontaining triphosphate were combined and lyophilized to yield7-[1-(4-methoxy-2-nitrophenyl)-2-methyl-propyloxy]methyl-7-deaza-2′-deoxyadenosine-5′-triphosphateWW6p087 as mixture of two diastereomers, which were separated by reversephase HPLC on a Perkin Elmer Aquapore OD-300 column (7 μm, 250×4.6 mm)to yield the single diastereomer WW6p087 ds1 (fast eluting) and WW6p087ds2 (slow eluting). Mobile phase: A, 100 mM triethylammonium acetate(TEAA) in water; B, 100 mM TEAA in water/CH₃CN (30:70).

Example 9 Synthesis of Chemically Cleavable Analogs Synthesis of5-(benzyloxy)methyl-2′-deoxyuridine-5′-triphosphate

5-(Benzyloxy)methyl-2′-deoxyuridine (dU.n1): Compound dU.x0 (381 mg,0.586 mmol) and benzyl alcohol (634 mg, 5.864 mmol) were heated neat at112° C. for 30 minutes under a nitrogen atmosphere. The mixture wascooled down to room temperature, dissolved in minimum amount ofdichloromethane, and purified by silica gel chromatography to yield5-(benzyloxy)methyl-2′-deoxyuridine dU.n1 (89 mg, 44%). It is noted thatdU.n1 is known (e.g., see Mel'nik et al., 1991, which is incorporatedherein by reference), but it was obtained in a different way thanreported here). ¹H NMR (400 MHz, DMSO-d₆): δ 11.40 (s, 1 H, D₂Oexchangeable, 3-NH), 7.94 (s, 1 H, H-6), 7.30 (m, 5 H, Ph-H), 6.17 (t, 1H, J=6.8 Hz, H-1′), 5.26 (d, J=4.2 Hz, 1 H, D₂O exchangeable, 3′-OH),5.04 (t, J=5.2 Hz, 1 H, D₂O exchangeable, 5′-OH), 4.49 (s, 2 H, PhCH₂),4.24 (m, 1 H, H-3′), 4.17 (m, 2 H, 5-CH₂a and 5-CH₂b), 3.79 (m, 1 H,H-4′), 3.57 (m, 2 H, H-5′a and H-5′b), 2.10 (m, 2 H, H-2′a and H-2′b);¹³C NMR (100 MHz, CD₃OD): δ 165.29 (C), 152.24 (C), 141.23 (CH), 139.62(C), 129.52 (CH), 129.05 (CH), 128.84 (CH), 112.44 (C), 89.04 (CH),86.73 (CH), 73.69 (CH₂), 72.27 (CH), 65.95 (CH₂), 62.92 (CH₂), 41.50(CH₂).

5-(Benzyloxy)methyl-2-deoxyuridine-5′-triphosphate (WW5p145): POCl₃ (8μL, 0.086 mmol) was added to a solution of compound dU.n1 (15 mg, 0.043mmol) and proton sponge (18 mg, 0.086 mmol) in trimethylphosphate (0.35mL) and the reaction was stirred at 0° C. under a nitrogen atmospherefor two hours. A solution of bis-tri-n-butylammonium pyrophosphate (237mg, 0.5 mmol) and tri-n-butylamine (100 μL) in anhydrous DMF (1 mL) wasadded. After 30 minutes of stirring, triethylammonium bicarbonate buffer(1 M, pH 7.5; 10 mL) was added. The reaction was stirred for one hour atroom temperature and then concentrated in vacuo. The residue wasdissolved in water (5 mL), filtered, and purified by anion exchangechromatography using a Q Sepharose FF column (2.5×10 cm) with a lineargradient of 25% acetonitrile/75% triethylammonium bicarbonate (TEAB,0.1M) to 25% acetonitrile/75% TEAB (1.5 M) over 240 min at 4.5 ml/min.The fractions containing triphosphate were combined and lyophilized toyield 5-(benzyloxy)methyl-2′-deoxyuridine-5′-triphosphate WW5p145. ¹HNMR (400 MHz, D₂O): δ 7.82 (s, 1 H, H-6), 7.26 (m, 5 H, Ph-H), 6.14 (t,J=6.8 Hz, 1 H, H-1′), 4.51 (m, 1 H, H-3′), 4.5 (s, 2 H, Ph-CH₂), 4.31 (2d, 2 H, 5-CH₂), 4.08 (m, 3 H, H-4′ and H-5′), 2.21 (m, 2 H, H-2′); ³¹PNMR (162 Hz, D₂O): 6-9.79 (d, J=19.4 Hz), −11.67 (d, J=21.0 Hz), −23.13(t, J=21.0 Hz).

Synthesis of5-(1-phenyl-2-methyl-propyloxy)methyl-2′-deoxyuridine-5′-triphosphate

5-(1-Phenyl-2-methyl-propyloxy)methyl-2′-deoxyuridine (dU.n2):CompounddU.x0 (0.331 g, 0.51 mmol) and 2-methyl-1-phenyl-1-propanol (1.238 g,8.24 mmol) were heated neat at 108-114° C. for one hour under a nitrogenatmosphere. The mixture was cooled down to room temperature, dissolvedin minimum amount of ethyl acetate, and purified by silica gelchromatography to yield5-(1-phenyl-2-methyl-propyloxy)methyl-2′-deoxyuridine dU.n2 (26 mg, 12%,1:1 mixture of diastereomers).3′,5′-O-Bis-(tert-butyldimethylsilyl)-5-(1-phenyl-2-methyl-propyloxy)methyl-2′-deoxy-uridine(77 mg, 24%, 1:1 mixture of diastereomers) and (3′ or5′)—O-(tert-butyldimethylsilyl)-5-(1-phenyl-2-methyl-propyloxy)methyl-2′-deoxyuridine(46 mg, 18%, 1:1 mixture of diastereomers) were also obtained from thereaction. ¹H NMR (400 MHz, DMSO-d₆) for dU.n2 (1:1 mixture ofdiastereomers): δ 11.31 (br s, 1 H, D₂O exchangeable, 3-NH), 7.77 (2 s,1 H, H-6), 7.29 (m, 5 H, Ph-H), 6.14 (m, 1 H, H-1′), 5.25 (d, J=4.4 Hz,1 H, D₂O exchangeable, 3′-OH), 4.98 (m, 1 H, D₂O exchangeable, 5′-OH),4.22 (m, 1 H, H-3′), 4.00 (m, 1 H, PhCH), 3.91 (m, 2 H, 5-CH₂a and5-CH₂b), 3.77 (m, 1 H, H-4′), 3.54 (m, 2 H, H-5′a and H-5′b), 2.06 (m, 2H, H-2′a and H-2′b), 1.83 (m, 2 H, CH(CH₃)₂), 0.88 (d, J=6.8 Hz, 3 H,CH₃), 0.66 (d, J=6.8 Hz, 3 H, CH₃).

¹H NMR (400 MHz, CDCl₃) for3′,5′-O-bis-(tert-butyldimethylsilyl)-5-(1-phenyl-2-methyl-propyloxy)methyl-2′-deoxyuridine(1:1 mixture of diastereomers): δ 9.07 and 9.06 (2 s, 1 H, 3-NH), 7.95and 7.53 (2 s, 1 H, H-6), 7.29 (m, 5 H, Ph-H), 6.29 (m, 1 H, H-1′), 4.24(m, 1 H, H-3′), 4.03 (m, 4 H, 5-CH₂a, 5-CH₂b, PhCH, and H-4′), 3.77 (ABdd, J=11.2 and 3.4 Hz, 1 H, H-5′a), 3.75 (AB dd, J=11.2 and 4.4 Hz, 1 H,H-5′b), 2.29 (m, 1 H, H-2′a), 1.98 (m, 1 H, H-2′b), 1.04 and 1.01 (2 d,J=6.4 and 6.8 Hz, 3 H, CH₃), 0.90 (s, 9 H, (CH₃)₃C), 0.89 and 0.88 (2 s,9 H, (CH₃)₃C), 0.74 and 0.73 (2 d, J=6.8 and 6.4 Hz, 3 H, CH₃) 0.10 and0.09 (2 s, 6 H, CH₃Si), 0.08 and 0.07 (2 s, 3 H, CH₃Si), 0.06 and 0.05(2 s, 3 H, (CH₃)₂Si); ¹³C NMR (100 MHz, CDCl₃) for3′,5′-O-bis-(tert-butyldimethylsilyl)-5-(1-phenyl-2-methyl-propyloxy)methyl-2′-deoxyuridine(1:1 mixture of diastereomers): δ 162.51 (C), 150.20 and 150.15 (C),140.83 and 140.79 (C), 137.28 and 137.19 (CH), 128.15 and 128.11 (CH),127.54 (CH), 127.45 (CH), 112.41 (C), 88.40 and 88.31 (CH), 87.83 and87.78 (CH), 85.38 and 85.30 (CH), 72.49 and 72.41 (CH), 63.64 and 63.57(CH₂), 63.22 (CH₂), 40.79 (CH₂), 34.82 and 34.79 (CH), 25.93 and 25.92(C(CH₃)₃), 25.76 and 25.72 (C(CH₃)₃), 19.20 and 19.17 (CH₃), 19.00(CH₃), 18.38 (C), 18.00 (C), −4.65 (CH₃), −4.80 (CH₃), −5.35 and −5.38(CH₃), −5.40 and −5.44 (CH₃).

¹H NMR (400 MHz, CDCl₃) for (3′ or5′)-O-(tert-butyldimethylsilyl)-5-(1-phenyl-2-methyl-propyloxy)methyl-2′-deoxyuridine(1:1 mixture of diastereomers): δ 9.09 (s, 1 H, 3-NH), 7.61 (s, 1 H,H-6), 7.28 (m, 5 H, Ph-H), 6.18 (m, 1 H, H-1′), 4.51 (m, 1 H, H-3′),4.09 (s, 2 H, 5-CH₂a, 5-CH₂b), 3.98 (d, J=7.2 Hz, 1 H, PhCH), 3.93 (m, 1H, H-4′), 3.90 (m, 1 H, H-5′a), 3.73 (m, 1 H, H-5′b), 2.50 (br, 1 H, 3′-or 5′-OH), 2.30 (m, 1 H, H-2′a), 1.98 (m, 2 H, H-2′b and CH(CH₃)₂), 1.01(d, J=6.4 Hz, 3 H, CH₃), 0.91 (s, 9 H, (CH₃)₃C), 0.74 (d, J=6.8 Hz, 3 H,CH₃), 0.10 (s, 3 H, CH₃Si), 0.07 and 0.06 (2 s, 3 H, CH₃Si).

5-(1-Phenyl-2-methyl-propyloxy)methyl-2-deoxyuridine-5′-triphosphate(WW5p143): POCl₃ (6 μL, 0.063 mmol) was added to a solution of compounddU.n2 (15 mg, 0.032 mmol) and proton sponge (14 mg, 0.063 mmol) intrimethylphosphate (0.4 mL) and the reaction was stirred at 0° C. undera nitrogen atmosphere for two hours. Additional POCl₃ (6 μL, 0.063 mmol)was added twice in one hour intervals. A solution ofbis-tri-n-butylammonium pyrophosphate (285 mg, 0.6 mmol) andtri-n-butylamine (120 μL) in anhydrous DMF (1.2 mL) was added. After 30minutes of stirring, triethylammonium bicarbonate buffer (1 M, pH 7.5;10 mL) was added. The reaction was stirred for one hour at roomtemperature and then concentrated in vacuo. The residue was dissolved inwater (5 mL), filtered, and purified by anion exchange chromatographyusing a Q Sepharose FF column (2.5×10 cm) with a linear gradient of 25%acetonitrile/75% triethylammonium bicarbonate (TEAB, 0.1M) to 25%acetonitrile/75% TEAB (1.5 M) over 240 min at 4.5 ml/min. The fractionscontaining triphosphate were combined and lyophilized to yield5-(1-phenyl-2-methyl-propyloxy)methyl-2′-deoxyuridine-5-′triphosphateWW5p143 (1:1 mixture of diastereomers); ³¹P NMR (162 Hz, D₂O): δ-10.88(m), −11.33 (m), −23.08 (m).

Synthesis of 5-(2-methylbenzyloxy)methyl-2′-deoxyuridine-5′-triphosphate

3′,5′-O-Bis-(tert-butyldimethylsilyl)-5-(2-methylbenzyloxy)methyl-2′-deoxyuridine(dU.n3) and (3′ or5′)-O-(tert-butyldimethylsilyl)-5-(2-methylbenzyloxy)methyl-2′-deoxyuridine(dU.n4:) Compound dU.x0 (0.438 g, 0.67 mmol) and 2-methylbenzyl alcohol(0.823 g, 6.74 mmol) were heated neat at 110° C. for 45 minutes under anitrogen atmosphere. The mixture was cooled down to room temperature andpurified by silica gel chromatography to yield3′,5′-O-bis-(tert-butyldimethylsilyl)-5-(2-methylbenzyloxy)methyl-2′-deoxyuridinedU.n3 (20 mg, 5%) and (3′ or5′)—O-(tert-butyldimethylsilyl)-5-(2-methylbenzyloxy)methyl-2′-deoxyuridinedU.n4 (43 mg, 14%). ¹H NMR (400 MHz, CDCl₃) for dU.n3: δ 8.42 (s, 1 H,NH), 7.66 (s, 1 H, H-6), 7.30 (m, 1 H, Ph-H), 7.18 (m, 3 H, Ph-H), 6.28(t, 1 H, H-1′), 4.59 (2 d, 2 H, Ph-CH₂), 4.38 (m, 1 H, H-3′), 4.27 (2 d,2 H, 5-CH₂), 3.94 (m, 1 H, H-4′), 3.75 (m, 2 H, H-5′), 2.34 (s, 3 H,CH₃), 2.26 (m, 1 H, H-2′a), 2.0 (m, 1 H, H-2′b), 0.89 and 0.90 (2 s, 18H, (CH₃)₃CSi), 0.09 and 0.08 (2 s, 6 H, (CH₃)₂Si), 0.07 and 0.06 (2 s, 6H, (CH₃)₂Si); ¹H NMR (400 MHz, CDCl₃) for dU.n4: δ 8.67 (s, 1 H, NH),7.73 (s, 1 H, H-6), 7.33 (m, 1 H, Ph-H), 7.22 (m, 3 H, Ph-H), 6.16 (t, 1H, J=6.4 Hz, H-1′), 4.6 (s, 2 H, Ph-CH₂), 4.46 (m, 1 H, H-3′), 4.34 (2d, 2 H, 5-CH₂), 3.92 (m, 1 H, H-4′), 3.81 (m, 1 H, H-5′a), 3.67 (m, 1 H,H-5′b), 2.35 (s, 3 H, CH₃), 2.30 (m, 1 H, H-2′a), 2.24 (m, 1 H, H-2′b),0.89 (1 s, 9 H, (CH₃)₃CSi), 0.07 (2 s, 6 H, (CH₃)₂Si).

5-(2-Methylbenzyloxy)methyl-2′-deoxyuridine (dU.n5): A solution ofcompound dU.n3 (20 mg, 0.034 mmol) in THF (2 mL) was treated withn-Bu₄NF (32 mg, 0.1 mmol) at room temperature for three hours.Separately a solution of compound dU.n4 (43 mg, 0.09 mmol) in THF (4 mL)was also treated with n-Bu₄NF (64 mg, 0.2 mmol) at room temperature forthree hours. The two reaction mixtures were combined, concentrated invacuo, and purified by silica gel column chromatography to yield5-(2-methylbenzyloxy)methyl-2′-deoxyuridine dU.n5 (17 mg, 38%). ¹H NMR(400 MHz, DMSO-d₆): δ 11.38 (s, 1 H, D₂O exchangeable, NH), 7.93 (s, 1H, H-6), 7.29 (m, 1 H, Ph-H), 7.15 (m, 3 H, Ph-H), 6.15 (t, 1 H, J=6.8Hz, H-1′), 5.24 (d, J=4.4 Hz, 1 H, D₂O exchangeable, 3′-OH), 5.02 (t, 1H, J=5.2 Hz, D₂O exchangeable, 5′-OH), 4.46 (s, 2 H, Ph-CH₂), 4.22 (m, 1H, H-3′), 4.16 (2 d, 2 H, 5-CH₂), 3.77 (m, 1 H, H-4′), 3.55 (m, 2 H,H-5′), 2.24 (s, 1 H, CH₃), 2.08 (m, 2 H, H-2′).

5-(2-Methylbenzyloxy)methyl-2′-deoxyuridine-5′-triphosphate (WW147):POCl₃ (8 μL, 0.083 mmol) was added to a solution of compound dU.n5 (15mg, 0.041 mmol) and proton sponge (18 mg, 0.083 mmol) intrimethylphosphate (0.35 mL) and the reaction was stirred at 0° C. undera nitrogen atmosphere for two hours. Additional POCl₃ (4 μL, 0.041 mmol)was added and the mixture was stirred for another two hours at 0° C. Asolution of bis-tri-n-butylammonium pyrophosphate (237 mg, 0.5 mmol) andtri-n-butylamine (100 μL) in anhydrous DMF (1 mL) was added. After 30minutes of stirring, triethylammonium bicarbonate buffer (1 M, pH 7.5;10 mL) was added. The reaction was stirred for one hour at roomtemperature and then concentrated in vacuo. The residue was dissolved inwater (5 mL), filtered, and purified by anion exchange chromatographyusing a Q Sepharose FF column (2.5×10 cm) with a linear gradient of 25%acetonitrile/75% triethylammonium bicarbonate (TEAB, 0.1M) to 25%acetonitrile/75% TEAB (1.5 M) over 240 min at 4.5 ml/min. The fractionscontaining triphosphate were combined and lyophilized to yield5-(2-methylbenzyloxy)methyl-2′-deoxyuridine-5′-triphosphate WW5p147. ¹HNMR (400 MHz, D₂O): δ 7.96 (s, 1 H, H-6), 7.32 (m, 1 H, Ph-H), 7.24 (m,3 H, Ph-H), 6.28 (t, J=6.8 Hz, 1 H, H-1′), 4.63 (m, 3 H, Ph-CH₂ andH-3′), 4.43 (2 d, 2 H, 5-CH₂), 4.20 (m, 3 H, H-4′ and H-5′), 2.36 (m, 2H, H-2′), 2.31 (s, 3 H, CH₃); ³¹P NMR (162 Hz, D₂O): δ-7.29 (m), −10.66(d, J=17.8 Hz), −21.4 (m).

Synthesis of5-(2-isopropylbenzyloxy)methyl-2′-deoxyuridine-5′-triphosphate

2-Isopropylbenzyl alcohol: To a solution of 1-bromo-2-isopropylbenzene(2.50 g, 12.56 mmol) in anhydrous THF (40 mL), 2,2′-dipyridyl (ca 2 mg)was added under nitrogen atmosphere (Zhi et al., 2003, which isincorporated herein by reference). The mixture was cooled down minus 78°C., and a solution of n-butyllithium (5.52 mL, 2.5 M in hexanes, 13.82mmol) was added dropwise via syringe within the period of ten minutes.Upon addition, the mixture was stirred for 30 minutes, then warmed up tominus 30° C., and a flow of formaldehyde (generated from 1.77 g ofparaformaldehyde by heating at 160° C.) was passed through the solutionuntil the deep red color disappeared completely. The mixture wasquenched with saturated ammonium chloride (5 mL), then poured into brine(15 mL). Organic layer was separated; aqueous layer was extracted twicewith dichloromethane (20 mL each); combined extracts were dried overanhydrous Na₂SO₄, evaporated, and purified by silica gel chromatographyto yield 2-isopropylbenzyl alcohol (1.11 g, 59%) as an oil. ¹H NMR (400MHz, CDCl₃): δ 7.30 (m, 3 H, Ph-H), 7.18 (m, 3 H, Ph-H), 4.75 (s, 2 H,CH ₂OH), 3.27 (sep, J=6.6 Hz, 1 H, CH(CH₃)₂), 1.53 (s, 1 H, CH₂OH), 1.26(d, J=6.6 Hz, 1 H, CH(CH ₃)₂).

5-(2-Isopropylbenzyloxy)methyl-2′-deoxyuridine (dU.n6): Compound dU.x0(0.331 g, 0.51 mmol) and 2-isopropylbenzyl alcohol (1.238 g, 8.24 mmol)were heated neat at 110-112° C. for 1.5 hours under nitrogen atmosphere.The mixture was cooled down to room temperature, dissolved in ethylacetate, and purified by silica gel chromatography to yield5-(2-isopropylbenzyloxy)methyl-2′-deoxyuridine dU.n6 (33 mg, 12%).3′,5′-O-bis-(tert-butyldimethylsilyl)-5-(2-isopropylbenzyloxy)methyl-2′-deoxyuridine(134 mg, 29%) and (3′ or5′)-O-(tert-butyldimethylsilyl)-5-(2-isopropylbenzyloxy)methyl-2′-deoxyuridine(98 mg, 26%) were also obtained from the reaction. ¹H NMR (400 MHz,DMSO-d₆) for dU.n6: δ 11.41 (s, 1H, D₂O exchangeable, NH), 7.94 (s, 1 H,H-6), 7.29 (m, 3 H, Ph-H), 7.14 (m, 1 H, Ph-H), 6.16 (t, 1 H, J=6.8 Hz,H-1′), 5.26 (t, J=4.4 Hz, 1 H, D₂O exchangeable, 5′-OH), 5.03 (t, J=5.2Hz, 1 H, D₂O exchangeable, 5′-OH), 4.51 (s, 2 H, CH₂O), 4.24 (m, 1 H,H-3′), 4.18 (AB d, J=12.7 Hz, 1 H, 5-CH₂a), 4.15 (AB d, J=12.7 Hz, 1 H,5-CH₂b), 3.78 (m, 1 H, H-4′), 3.56 (m, 2 H, H-5′a and H-5′b), 3.15 (sep,J=6.8 Hz, 1 H, CH(CH₃)₂), 2.09 (m, 2 H, H-2′a and H-2′b), 1.15 (d, J=6.8Hz, 6 H, CH(CH ₃)₂); ¹³C NMR (100 MHz, CD₃OD) for dU.n6: δ 163.77 (C),150.77 (C), 147.85 (C), 139.85 (CH), 134.29 (C), 129.41 (CH), 128.19(CH), 125.19 (CH), 125.03 (CH), 110.97 (C), 87.56 (CH), 85.22 (CH),70.85 (CH), 70.35 (CH₂), 64.29 (CH₂), 61.47 (CH₂), 40.01 (CH₂), 28.39(CH), 23.09 (CH₃).

¹H NMR (400 MHz, CDCl₃) for3′,5′-O-bis-(tert-butyldimethylsilyl)-5-(2-isopropylbenzyloxy)methyl-2′-deoxyuridine:δ 9.60 (s, 1 H, 3-NH), 7.64 (s, 1 H, H-6), 7.28 (m, 3 H, Ph-H), 7.28(dt, J=7.1 and 1.8 Hz, 1 H, Ph-H), 6.29 (dd, J=7.7 and 5.9 Hz, 1 H,H-1′), 4.64 (s, 2 H, CH₂O), 4.38 (m, 1 H, H-3′), 4.31 (AB d, J=12.7 Hz,1 H, 5-CH₂a), 4.27 (AB d, J=12.7 Hz, 1 H, 5-CH₂b), 3.93 (m, 1 H, H-4′),3.74 (m, 2 H, H-5′a and H-5′b), 3.24 (sep, J=6.8 Hz, 1 H, CH(CH₃)₂),2.28 (m, 1 H, H-2′a), 2.00 (m, 1 H, H-2′b), 1.23 (d, J=6.8 Hz, 6 H,CH(CH ₃)₂), 0.89 (s, 9 H, (CH₃)₃C), 0.88 (s, 9 H, (CH₃)₃C), 0.09 (s, 3H, CH₃Si), 0.07 (s, 3 H, CH₃Si), 0.06 (s, 3 H, CH₃Si), 0.05 (s, 3 H,CH₃Si); ¹³C NMR (100 MHz, CDCl₃) for3′,5′-O-bis-(tert-butyldimethylsilyl)-5-(2-isopropylbenzyloxy)methyl-2′-deoxyuridine:δ 162.94 (C), 150.29 (C), 147.74 (C), 138.11 (CH), 134.27 (C), 129.41(CH), 128.40 (CH), 125.54 (CH), 125.38 (CH), 111.90 (C), 87.81 (CH),85.28 (CH), 72.24 (CH), 71.08 (CH₂), 64.44 (CH₂), 62.99 (CH₂), 41.08(CH₂), 28.63 (CH), 25.92 (C(CH₃)₃), 25.74 (C(CH₃)₃), 23.99 (CH₃), 18.36(C), 17.98 (C), −4.67 (CH₃), −4.84 (CH₃), −5.44 (CH₃), −5.52 (CH₃).

¹H NMR (400 MHz, DMSO-d₆) for (3′ or5′)-O-(tert-butyldimethylsilyl)-5-(2-isopropylbenzyloxy)methyl-2′-deoxyuridine:δ 11.42 (s, 1 H, D₂O exchangeable, NH), 7.89 (s, 1 H, H-6), 7.28 (m, 3H, Ph-H), 7.12 (m, 1 H, Ph-H), 6.14 (t, 1 H, J=6.8 Hz, H-1′), 5.07 (t,J=5.2 Hz, 1 H, D₂O exchangeable, 5′-OH), 4.51 (s, 2 H, CH₂O), 4.41 (m, 1H, H-3′), 4.31 (AB d, J=12.7 Hz, 1 H, 5-CH₂a), 4.15 (AB d, J=12.7 Hz, 1H, 5-CH₂b), 3.76 (m, 1 H, H-4′), 3.56 (m, 2 H, H-5′a and H-5′b), 3.14(sep, J=6.8 Hz, 1 H, CH(CH₃)₂), 2.16 (m, 1 H, H-2′a), 2.09 (m, 1 H,H-2′b), 1.14 (d, J=6.8 Hz, 6 H, CH(CH ₃)₂), 0.86 (s, 9 H, (CH₃)₃C), 0.06(s, 6 H, (CH₃)₂Si); ¹³C NMR (100 MHz, CDCl₃) for (3′ or5′)-O-(tert-butyldimethylsilyl)-5-(2-isopropylbenzyloxy)methyl-2′-deoxy-uridine:δ 162.77 (C), 150.30 (C), 147.76 (C), 138.79 (CH), 134.26 (C), 129.32(CH), 128.54 (CH), 125.60 (CH), 125.48 (CH), 111.92 (C), 87.77 (CH),87.14 (CH), 71.72 (CH), 71.03 (CH₂), 64.40 (CH₂), 61.98 (CH₂), 40.68(CH₂), 28.68 (CH), 25.71 (C(CH₃)₃), 23.99 (CH₃), 17.93 (C), −4.73 (CH₃),−4.88 (CH₃).

Compounds3′,5′-O-bis-(tert-butyldimethylsilyl)-5-(2-isopropylbenzyl-oxy)methyl-2′-deoxyuridine(134 mg, 0.22 mmol) and (3′ or5′)-O-(tert-butyldimethylsilyl)-5-(2-isopropylbenzyloxy)methyl-2′-deoxyuridine(98 mg, 0.19 mmol) were dissolved in THF (5 mL), and a solution oftetra-n-butylammonium fluoride trihydrate (323 mg, 1.05 mmol) in THF (2mL) was added. The mixture was stirred at room temperature for one hour,then concentrated in vacuo and purified by silica gel columnchromatography to give 5-(2-isopropylbenzyloxy)methyl-2′-deoxyuridinedU.n6 (108 mg, 67%).

5-(2-Isopropylbenzyloxy)methyl-2′-deoxyuridine-5′triphosphate (WW5p149):POCl₃ (15 μL, 0.164 mmol) was added to a solution of compound dU.n6 (32mg, 0.082 mmol) and proton sponge (35 mg, 0.164 mmol) intrimethylphosphate (0.5 mL) and the reaction was stirred at 0° C. undera nitrogen atmosphere for 2 hours. A solution of bis-tri-n-butylammoniumpyrophosphate (356 mg, 0.75 mmol) and tri-n-butylamine (150 μL) inanhydrous DMF (1.5 mL) was added. After 30 minutes of stirring,triethylammonium bicarbonate buffer (1 M, pH 7.5; 10 mL) was added. Thereaction was stirred for one hour at room temperature and thenconcentrated in vacuo. The residue was dissolved in a mixture of water(5 mL) and acetonitrile (5 mL), filtered, and purified by anion exchangechromatography using a Q Sepharose FF column (2.5×10 cm) with a lineargradient of 25% acetonitrile/75% triethylammonium bicarbonate (TEAB,0.1M) to 25% acetonitrile/75% TEAB (1.5 M) over 240 min at 4.5 ml/min.The fractions containing triphosphate were combined and lyophilized toyield 5-(2-isopropylbenzyloxy)methyl-2′-deoxyuridine-5′-triphosphateWW5p149. ¹H NMR (400 MHz, D₂O): δ 7.98 (s, 1 H, H-6), 7.36 (m, 3 H,Ph-H), 7.21 (m, 1 H, Ph-H), 6.27 (t, J=6.8 Hz, 1 H, H-1′), 4.64 (m, 3 H,Ph-CH₂ and H-3′), 4.43 (AB d, 1 H, J=12 Hz, 5-CH₂a), 4.39 (AB d, 1 H,J=12 Hz, 5-CH₂b), 4.25 (m, 3 H, H-4′ and H-5′), 2.34 (m, 2 H, H-2′),1.15 (d, 3 H, J=6.8 Hz, CH₃); ³¹P NMR (162 Hz, D₂O): 6-5.11 (d, J=21.0Hz), −10.5 (d, J=19.4 Hz), −20.94 (t, J=21.0 Hz).

Synthesis of5-(2-tert-butylbenzyloxy)methyl-2′-deoxyuridine-5′-triphosphate

1-Bromo-2-tert-butylbenzene: To a solution of 2-tert-butylaniline (7.46g, 50 mmol 15.6 mL) in hydrobromic acid (40% w/w, 15 mL) cooled at <5°C. (ice/salt bath), a solution of 7.55 g (0.11 mol) of sodium nitrite in10 mL of water was added at a rate that the temperature did not exceed10° C. (ca two hour addition time). When the diazotization wascompleted, 0.20 g of copper powder was added. (CAUTION: the solution wasrefluxed very cautiously because of vigorous gas evolution!). When thevigorous evolution of nitrogen subsided, the system was kept at 50° C.for 30 minutes and was then diluted with 80 mL of water and extractedthree times with diethyl ether (100 mL each). The organic layer waswashed with 10% solution of KOH; dried over Na₂SO₄, concentrated invacuo, and purified by chromatography on silica gel chromatography. Theproduct obtained was further distilled at 85° C. (3 mm Hg) to yield1-bromo-2-tert-butylbenzene (2.88 g, 27%). ¹H NMR (400 MHz, CDCl₃): δ7.58 (m, 1 H, Ph-H), 7.45 (m, 1 H, Ph-H), 7.24 (m, 1 H, Ph-H), 7.02 (m,1 H, Ph-H), 1.51 (s, 9 H, C(CH₃)₃).

2-tert-Butylbenzyl alcohol: To a solution of 1-bromo-2-tert-butylbenzene(2.88 g, 13.51 mmol) in anhydrous THF (45 mL) 2,2′-dipyridyl (ca 10 mg)was added under a nitrogen atmosphere. The mixture was cooled down minus78° C., and a solution of n-butyllithium (2.5 M in hexanes, 5.94 mL,14.86 mmol) was added dropwise via syringe within the period of tenminutes. Upon addition, the mixture was stirred for 30 minutes, thenwarmed up to minus 30° C., and a flow of formaldehyde (generated from1.91 g of paraformaldehyde by heating at over 160° C.) was passedthrough the solution until the deep red color disappeared completely.The mixture was quenched with saturated ammonium chloride solution (5mL), then poured into a mixture of dichloromethane (100 mL) and water(50 mL). The organic phase was separated and the aqueous phase wasextracted with dichloromethane (20 mL) twice. The combined organic phasewas dried over anhydrous Na₂SO₄, concentrated in vacuo, and purified bysilica gel chromatography to yield 2-tert-butylbenzyl alcohol (1.67 g,75%) as an oil. ¹H NMR (400 MHz, CDCl₃): δ 7.50 (m, 1 H, Ph-H), 7.41 (m,1 H, Ph-H), 7.24 (m, 2 H, Ph-H), 4.93 (d, J=4.8 Hz, 2 H, CH ₂OH), 1.54(br, 1 H, CH₂OH), 1.43 (s, 1 H, C(CH₃)₃).

3′,5′-O-Bis-(tert-butyldimethylsilyl)-5-(2-tert-butylbenzyloxy)methyl-2′-deoxyuridine(dU.n7) and (3′ or5′)-O-(tert-butyldimethylsilyl)-5-(2-tert-butylbenzyloxy)methyl-2′-deoxyuridine(dU.n8): Compound dU.x0 (230 mg, 0.36 mmol) and 2-tert-butylbenzylalcohol (0.49 g, 3.60 mmol) were heated neat at 118-122° C. for one hourunder a nitrogen atmosphere. The mixture was cooled down to roomtemperature, dissolved in minimum amount of ethyl acetate, and purifiedby silica gel chromatography to yield3′,5′-O-bis-(tert-butyldimethylsilyl)-5-(2-tert-butylbenzyloxy)methyl-2′-deoxyuridinedU.n7 (32 mg, 17%) and (3′ or5′)-O-(tert-butyldimethylsilyl)-5-(2-tert-butylbenzyloxy)methyl-2′-deoxyuridinedU.n8 (28 mg, 19%). ¹H NMR (400 MHz, CDCl₃) for dU.n7: δ 9.28 (s, 1 H,3-NH), 7.77 (s, 1 H, H-6), 7.43 (m, 2 H, Ph-H), 7.25 (m, 2 H, Ph-H),6.16 (t, J=6.6 Hz, 1 H, H-1′), 4.84 (AB d, J=11.2 Hz, 1 H, 5-CH₂a), 4.81(AB d, J=11.2 Hz, 1 H, 5-CH₂b), 4.40 (m, 1 H, H-3′), 4.36 (s, 2 H,CH₂O), 3.91 (m, 1 H, H-4′), 3.76 (AB d, J=12.0 Hz, 1 H, H-5′a), 3.64 (ABd, J=12.0 Hz, 1 H, H-5′b), 2.27 (m, 2 H, H-2′a and H-2′b), 1.40 (s, 9 H,C(CH₃)₃), 0.89 (s, 9H, SiC(CH₃)₃), 0.88 (s, 9 H, (CH₃)₃CSi), 0.09 (s, 3H, CH₃Si), 0.07 (s, 3 H, CH₃Si), 0.06 (s, 3 H, CH₃Si), 0.05 (s, 3 H,CH₃Si); ¹H NMR (400 MHz, DMSO-d₆) for dU.n8: δ 11.42 (s, 1 H, D₂Oexchangeable, NH), 7.93 (s, 1 H, H-6), 7.42 (m, 1 H, Ph-H), 7.35 (m, 1H, Ph-H), 7.18 (m, 2 H, Ph-H), 6.15 (t, 1 H, J=6.8 Hz, H-1′), 5.08 (t,J=5.2 Hz, 1 H, D₂O exchangeable, 5′-OH), 4.66 (s, 2 H, CH₂O), 4.41 (m, 1H, H-3′), 4.26 (AB d, J=11.6 Hz, 1 H, 5-CH₂a), 4.21 (AB d, J=11.6 Hz, 1H, 5-CH₂b), 3.78 (m, 1 H, H-4′), 3.56 (m, 2 H, H-5′a and H-5′b), 2.18(m, 2 H, H-2′a and H-2′b), 1.34 (s, 9 H, (CH ₃)₃C), 0.85 (s, 9 H,(CH₃)₃CSi), 0.07 (s, 6 H, (CH₃)₂Si).

5-(2-tert-butylbenzyloxy)methyl-2′-deoxyuridine (dU.n9): Compounds dU.n7(32 mg, 0.050 mmol) and dU.n8 (27 mg, 0.052 mmol) were dissolved in THF(4 mL), and a solution of tetra-n-butylammonium fluoride trihydrate (65mg, 0.204 mmol) in THF (2 mL) was added. The mixture was stirred at roomtemperature for one hour, then concentrated in vacuo and purified bysilica gel column chromatography to give5-(2-tert-butylbenzyloxy)methyl-2′-deoxyuridine dU.n9 (108 mg, 67%). ¹HNMR (400 MHz, CD₃OD): δ 8.10 (s, 1 H, H-6), 7.46 (m, 1 H, Ph-H), 7.39(m, 1 H, Ph-H), 7.20 (m, 1 H, Ph-H), 6.28 (t, 1 H, J=6.6 Hz, H-1′), 4.79(s, 2 H, CH₂O), 4.37 (m, 3 H, 5-CH₂a, 5-CH₂b, and H-3′), 3.92 (m, 1 H,H-4′), 3.76 (AB dd, J=12.4 and 3.2 Hz, 1 H, H-5′a), 3.70 (AB dd, J=12.4and 3.6 Hz, 1 H, H-5′a, H-5′b), 2.22 (m, 2 H, H-2′a and H-2′b), 1.39 (s,9 H, C(CH₃)₃).

5-(2-tert-butylbenzyloxy)methyl-2′-deoxyuridine-5′-triphosphate(WW6p024): POCl₃ (10 μL, 0.11 mmol) was added to a solution of compounddU.n9 (30 mg, 0.074 mmol) and proton sponge (32 mg, 0.15 mmol) intrimethylphosphate (0.4 mL) and the reaction was stirred at 0° C. undera nitrogen atmosphere for two hours. A solution ofbis-tri-n-butylammonium pyrophosphate (285 mg, 0.6 mmol) andtri-n-butylamine (120 μL) in anhydrous DMF (1.2 mL) was added. After 30minutes of stirring, triethylammonium bicarbonate buffer (1 M, pH 7.5;10 mL) was added. The reaction was stirred for one hour at roomtemperature and then concentrated in vacuo. The residue was dissolved ina mixture of water (5 mL) and acetonitrile (5 mL), filtered, andpurified by anion exchange chromatography using a Q Sepharose FF column(2.5×10 cm) with a linear gradient of 25% acetonitrile/75%triethylammonium bicarbonate (TEAB, 0.1M) to 25% acetonitrile/75% TEAB(1.5 M) over 240 min at 4.5 ml/min. The fractions containingtriphosphate were combined and lyophilized to yield5-(2-tert-butylbenzyloxy)methyl-2′-deoxyuridine-5′-triphosphate WW6p024.¹H NMR (400 MHz, D₂O): δ 7.89 (s, 1 H, H-6), 7.34 (d, 1 H, J=1.6 Hz,Ph-H), 7.32 (d, 1 H, J=2.0 Hz, Ph-H), 7.15 (m, 2 H, Ph-H), 6.17 (t,J=6.8 Hz, 1 H, H-1′), 4.64 (2 d, 2 H, Ph-CH₂), 4.48 (m, 1 H, H-3′), 4.32(2 d, 2 H, 5-CH₂), 4.04 (m, 3 H, H-4′ and H-5′), 2.23 (m, 2 H, H-2′),1.20 (s, 9 H, C(CH₃)₃); ³¹P NMR (162 Hz, D₂O): δ-11.76 (m), −12.41 (d,J=19.4 Hz), −24.0 (t, J=19.4 Hz).

Synthesis of 5-(2-phenylbenzyloxy)methyl-2′-deoxyuridine-5′-triphosphate

5-(2-Phenylbenzyloxy)methyl-2′-deoxyuridine (dU.n10): Compound dU.x0(0.916 g, 1.41 mmol) and 2-biphenylmethanol (2.596 g, 14.10 mmol) wereheated neat at 118-122° C. for 1.5 hours under a nitrogen atmosphere.The mixture was cooled down to room temperature, dissolved in ethylacetate, and purified by silica gel chromatography to yield5-(2-phenylbenzyloxy)methyl-2′-deoxyuridine dU.n10 (87 mg, 15%).3′,5′-O-Bis-(tert-butyldimethylsilyl)-5-(2-phenylbenzyloxy)methyl-2′-deoxyuridine(134 mg, 19%) and (3′ or5′)-O-(tert-butyldimethylsilyl)-5-(2-phenylbenzyloxy)methyl-2′-deoxyuridine(263 mg, 36%) were also obtained from the reaction. ¹H NMR (400 MHz,DMSO-d₆) for dU.n10: δ 11.40 (s, 1 H, D₂O exchangeable, NH), 7.91 (s, 1H, H-6), 7.39 (m, 9 H, Ph-H), 6.14 (t, 1 H, J=6.8 Hz, H-1′), 5.25 (t,J=4.4 Hz, 1 H, D₂O exchangeable, 5′-OH), 5.02 (t, J=5.2 Hz, 1 H, D₂Oexchangeable, 5′-OH), 4.33 (s, 2 H, CH₂O), 4.21 (m, 1 H, H-3′), 4.15 (ABd, J=11.6 Hz, 1 H, 5-CH₂a), 4.08 (AB d, J=12.7 Hz, 1 H, 5-CH₂b), 3.78(m, 1 H, H-4′), 3.55 (m, 2 H, H-5′a and H-5′b), 2.05 (m, 2 H, H-2′a andH-2′b); ¹H NMR (400 MHz, CDCl₃) for3′,5′-O-bis-(tert-butyldimethylsilyl)-5-(2-phenylbenzyloxy)methyl-2′-deoxyuridine:δ 8.75 (s, 1 H, 3-NH), 7.63 (s, 1 H, H-6), 7.37 (m, 9 H, Ph-H), 6.29(dd, J=7.6 and 6.0 Hz, 1 H, H-1′), 4.49 (s, 2 H, CH₂O), 4.39 (m, 1 H,H-3′), 4.24 (AB d, J=12.7 Hz, 1 H, 5-CH₂a), 4.19 (AB d, J=12.7 Hz, 1 H,5-CH₂b), 3.96 (m, 1 H, H-4′), 3.76 (m, 2 H, H-5′a and H-5′b), 2.28 (m, 1H, H-2′a), 2.03 (m, 1 H, H-2′b), 0.91 (s, 9 H, (CH₃)₃C), 0.89 (s, 9 H,(CH₃)₃C), 0.09 (s, 3 H, CH₃Si), 0.06 (s, 3 H, CH₃Si), 0.05 (s, 3 H,CH₃Si), 0.02 (s, 3 H, CH₃Si): ¹H NMR (400 MHz, DMSO-d₆) for (3′ or5′)-O-(tert-butyldimethylsilyl)-5-(2-phenylbenzyloxy)methyl-2′-deoxyuridine:δ 11.42 (s, 1 H, D₂O exchangeable, NH), 7.86 (s, 1 H, H-6), 7.39 (m, 9H, Ph-H), 6.13 (t, 1 H, J=6.8 Hz, H-1′), 5.08 (t, J=5.2 Hz, 1 H, D₂Oexchangeable, 5′-OH), 4.39 (m, 1 H, H-3′), 4.33 (s, 2 H, CH₂O), 4.15 (ABd, J=11.6 Hz, 1 H, 5-CH₂a), 4.09 (AB d, J=11.6 Hz, 1 H, 5-CH₂b), 3.77(m, 1 H, H-4′), 3.54 (m, 2 H, H-5′a and H-5′b), 2.08 (m, 2 H, H-2′a andH-2′b), 0.87 (s, 9 H, (CH₃)₃C), 0.07 (2 s, 6 H, (CH₃)₂Si).

5-(2-Phenylbenzyloxy)methyl-2′-deoxyuridine-5′-triphosphate (WW6p010):POCl₃ (9 μL, 0.1 mmol) was added to a solution of compound dU.n10 (28mg, 0.066 mmol) and proton sponge (28 mg, 0.13 mmol) intrimethylphosphate (0.4 mL) and the reaction was stirred at 0° C. undera nitrogen atmosphere for two hours. A solution ofbis-tri-n-butylammonium pyrophosphate (285 mg, 0.6 mmol) andtri-n-butylamine (120 μL) in anhydrous DMF (1.2 mL) was added. After 30minutes of stirring, triethylammonium bicarbonate buffer (1 M, pH 7.5;10 mL) was added. The reaction was stirred for one hour at roomtemperature and then concentrated in vacuo. The residue was dissolved ina mixture of water (6 mL) and acetonitrile (4 mL), filtered, andpurified by anion exchange chromatography using a Q Sepharose FF column(2.5×10 cm) with a linear gradient of 25% acetonitrile/75%triethylammonium bicarbonate (TEAB, 0.1M) to 25% acetonitrile/75% TEAB(1.5 M) over 240 min at 4.5 ml/min. The fractions containingtriphosphate were combined and lyophilized to yield5-(2-phenylbenzyloxy)methyl-2′-deoxyuridine-5′-triphosphate WW6p010. ¹HNMR (400 MHz, D₂O): δ 7.77 (s, 1 H, H-6), 7.56 (m, 1 H, Ph-H), 7.44 (m,2 H, Ph-H), 7.39 (m, 3 H, Ph-H), 7.27 (m, 3 H, Ph-H), 6.25 (t, J=6.8 Hz,1 H, H-1′), 4.62 (m, 1 H, H-3′), 4.41 (AB d, 1 H, J=11.2 Hz, 5-CH₂a),4.36 (AB d, 1 H, J=12 Hz, 5-CH₂b), 4.24 (m, 1 H, H-4′), 4.17 (m, 2 H,H-5′), 2.30 (m, 2 H, H-2′); ³¹P NMR (162 Hz, D₂O): 6-5.59 (d, J=19.4Hz), −10.6 (d, J=19.4 Hz), −21.04 (t, J=19.4 Hz).

Synthesis of5-(2,6-dimethylbenzyloxy)methyl-2′-deoxyuridine-5′-triphosphate

2,6-dimethylbenzyl alcohol: 2,6-Dimethylbenzyl alcohol was preparedaccording to Beaulieu et al. (2000, which is incorporated herein byreference), but was unsuccessful, so a different reducing agent wasused. To a suspension of 2,6-dimethylbenzoic acid (1.00 g, 6.65 mmol) inanhydrous THF (10 mL) a solution of BH₃(SMe₂) in THF was cautiouslyadded under nitrogen atmosphere. The mixture was heated at reflux for 16hours, then quenched with saturated ammonium chloride (5 mL) and 2 M HCl(10 mL). (CAUTION: vigorous gas evolution!). Organic layer wasseparated; aqueous layer was extracted three times with ethyl acetate(45 mL each); combined extracts were washed twice with saturated sodiumbicarbonate (20 mL each), dried over anhydrous Na₂SO₄, evaporated, andpurified by silica gel chromatography to yield 2,6-dimethylbenzylalcohol (0.50 g, 51%) as an white solid. ¹H NMR (400 MHz, CDCl₃): δ 7.08(m, 3 H, Ph-H), 4.70 (s, 2 H, Ph-CH₂), 4.05 (br s, 1 H, OH), 2.40 (s, 6H, CH₃).

3′,5′-O-Bis-(tert-butyldimethylsilyl)-5-(2,6-dimethylbenzyloxy)methyl-2′-deoxyuridine(dU.n11): Compound dU.x0 (230 mg, 0.36 mmol) and 2,6-dimethylbenzylalcohol (0.49 g, 3.60 mmol) were heated neat at 118-122° C. for one hourunder a nitrogen atmosphere. The mixture was cooled down to roomtemperature, dissolved in minimum amount of ethyl acetate, and purifiedby silica gel chromatography to yield3′,5′-O-bis-(tert-butyldimethylsilyl)-5-(2,6-dimethylbenzyloxy)methyl-2′-deoxyuridinedU.n11 (170 mg, 80%). ¹H NMR (400 MHz, CDCl₃): δ 8.94 (s, 1 H, 3-NH),7.63 (s, 1 H, H-6), 7.07 (m, 3 H, Ph-H), 6.28 (dd, J=7.6 and 5.6 Hz, 1H, H-1′), 4.63 (s, 2 H, CH₂O), 4.38 (m, 1 H, H-3′), 4.32 (AB d, J=11.6Hz, 1 H, 5-CH₂a), 4.27 (AB d, J=11.6 Hz, 1 H, 5-CH₂b), 3.92 (m, 1 H,H-4′), 3.74 (m, 2 H, H-5′a and H-5′b), 2.39 (s, 6 H, 2 CH₃), 2.24 (m, 1H, H-2′a), 2.00 (m, 1 H, H-2′b), 0.90 (s, 9 H, (CH₃)₃C), 0.89 (s, 9 H,(CH₃)₃C), 0.11 (s, 3 H, CH₃Si), 0.09 (s, 3 H, CH₃Si), 0.08 (s, 3 H,CH₃Si), 0.06 (s, 3 H, CH₃Si).

5-(2,6-Dimethylbenzyloxy)methyl-2′-deoxyuridine (dU.n12): To a solutionof compound dU.n11 (131 mg, 0.22 mmol) in THF (4 mL), a solution oftetra-n-butylammonium fluoride trihydrate (171 mg, 0.54 mmol) in THF (2mL) was added. The mixture was stirred at room temperature for 30minutes, then concentrated in vacuo and purified by silica gel columnchromatography to give 5-(2,6-dimethylbenzyloxy)methyl-2′-deoxyuridinedU.n12 (108 mg, 67%). ¹H NMR (400 MHz, CD₃OD): δ 8.08 (s, 1 H, H-6),7.02 (m, 3 H, Ph-H), 6.26 (t, 1 H, J=6.8 Hz, H-1′), 4.61 (s, 2 H, CH₂O),4.38 (m, 1 H, H-3′), 4.33 (AB d, J=11.6 Hz, 1 H, 5-CH₂a), 4.15 (AB d,J=11.6 Hz, 1 H, 5-CH₂b), 3.91 (m, 1 H, H-4′), 3.77 (AB dd, J=12.0 and3.2 Hz, 1 H, H-5′a), 3.70 (AB dd, J=12.0 and 3.6 Hz, 1 H, H-5′a, H-5′b),2.36 (s, 6 H, CH₃), 2.24 (m, 2 H, H-2′a and H-2′b); ¹³C NMR (100 MHz,CD₃OD): δ 163.80 (C), 150.76 (C), 140.85 (CH), 137.81 (C), 133.73 (C),127.81 (CH), 127.72 (CH), 110.82 (C), 87.56 (CH), 85.22 (CH), 70.74(CH), 66.33 (CH₂), 64.47 (CH₂), 61.38 (CH₂), 40.02 (CH₂), 18.36 (CH₃).

5-(2,6-Dimethylbenzyloxy)methyl-2-deoxyuridine-5′-triphosphate(WW6p015): POCl₃ (11 μL, 0.12 mmol) was added to a solution of compounddU.n12 (30 mg, 0.08 mmol) and proton sponge (34 mg, 0.16 mmol) intrimethylphosphate (0.4 mL) and the reaction was stirred at 0° C. undera nitrogen atmosphere for two hours. A solution ofbis-tri-n-butylammonium pyrophosphate (285 mg, 0.5 mmol) andtri-n-butylamine (120 μL) in anhydrous DMF (1.2 mL) was added. After 30minutes of stirring, triethylammonium bicarbonate buffer (1 M, pH 7.5;10 mL) was added. The reaction was stirred for one hour at roomtemperature and then concentrated in vacuo. The residue was dissolved ina mixture of water (5 mL) and acetonitrile (5 mL), filtered, andpurified by anion exchange chromatography using a Q Sepharose FF column(2.5×10 cm) with a linear gradient of 25% acetonitrile/75%triethylammonium bicarbonate (TEAB, 0.1M) to 25% acetonitrile/75% TEAB(1.5 M) over 240 min at 4.5 ml/min. The fractions containingtriphosphate were combined and lyophilized to yield5-(2,6-dimethylbenzyloxy)methyl-2′-deoxyuridine-5′-triphosphate WW6p015.¹H NMR (400 MHz, D₂O): δ 8.0 (s, 1 H, H-6), 7.16 (m, 1 H, Ph-H), 7.07(m, 3 H, Ph-H), 6.30 (t, 1 H, J=7.2 Hz, H-1′), 4.64 (m, 3 H, Ph-CH₂ andH-3′), 4.47 (AB d, 1 H, J=7.2 Hz, 5-CH₂a), 4.40 (AB d, 1 H, J=7.2 Hz,5-CH₂b), 4.20 (m, 3 H, H-4′ and H-5′), 2.38 (m, 2 H, H-2′), 2.33 (s, 6H, CH₃); ³¹P NMR (162 Hz, D₂O): 6-8.94 (d, J=19.4 Hz), −10.78 (d, J=19.4Hz), −22.08 (d, J=19.4 Hz).

Synthesis of 5-(3-phenyl-2-propenyloxy)methyl-2′-deoxyuridine andreaction to its 5′-triphosphate

3′,5′-O-Bis-(tert-butyldimethylsilyl)-5-(3-phenyl-2-propenyloxy)methyl-2′-deoxyuridine(dU.n13): Compound dU.x0 (500 mg, 0.77 mmol) and cinnamyl alcohol (331mg, 2.17 mmol) was heated neat at 104° C. for one hour under a nitrogenatmosphere. The mixture was cooled down to room temperature, dissolvedin minimum amount of ethyl acetate, and purified by silica gelchromatography to yield3′,5′-O-bis-(tert-butyldimethylsilyl)-5-(3-phenyl-2-propenyloxy)methyl-2′-deoxyuridinedU.n13 (169 mg, 36%). ¹H NMR (400 MHz, CDCl₃): δ 8.32 (s, 3-NH), 7.68(s, 1 H, H-6), 7.31 (m, 5 H, Ph-H), 6.64 (d, J=16.0 Hz, 1 H, ═CH), 6.28(m, 2 H, H-1′ and ═CH), 4.40 (m, 1 H, H-3′), 4.28 (m, 2 H, 5-CH₂a and5-CH₂b), 4.23 (m, 2 H, CH₂O), 3.94 (m, 1 H, H-4′), 3.80 (AB dd, J=11.2and 3.2 Hz, 1 H, H-5′a), 3.75 (AB dd, J=11.2 and 3.2 Hz, 1 H, H-5′b),2.27 (m, 1 H, H-2′a), 2.01 (m, 1 H, H-2′b), 0.90 (s, 9 H, (CH₃)₃C), 0.88(s, 9 H, (CH₃)₃C), 0.08 (s, 3 H, CH₃Si), 0.07 (s, 3 H, CH₃Si), 0.05 (2s, 6 H, (CH₃)₂Si); ¹³C NMR (100 MHz, CDCl₃): δ 162.56 (C), 150.04 (C),138.34 (CH), 136.58 (C), 133.06 (CH), 128.54 (CH), 127.76 (CH), 126.55(CH), 125.60 (CH), 111.80 (C), 87.92 (CH), 85.28 (CH), 72.27 (CH), 71.67(CH₂), 64.40 (CH₂), 62.99 (CH₂), 41.24 (CH₂), 25.96 (C(CH₃)₃), 25.76(C(CH₃)₃), 18.42 (C), 18.01 (C), −4.65 (CH₃), −4.82 (CH₃), −5.40 (CH₃),−5.50 (CH₃).

5-(3-Phenyl-2-propenyloxy)methyl-2′-deoxyuridine (dU.n14): To a solutionof compound dU.n13 (138 mg, 0.23 mmol) in THF (2 mL) a solution oftetra-n-butylammonium fluoride trihydrate (321 mg, 0.73 mmol) in THF (2mL) was added. The mixture was stirred at room temperature for one hour,then concentrated in vacuo and purified by silica gel columnchromatography to give 5-(3-phenyl-2-propenyloxy)methyl-2′-deoxyuridinedU.n14 (65 mg, 76%) as a waxy solid. ¹H NMR (400 MHz, DMSO-d₆): δ 11.39(br s, 1 H, D₂O exchangeable, NH), 7.93 (s, 1 H, H-6), 7.44 (m, 2 H,Ph-H), 7.32 (m, 2 H, Ph-H), 7.23 (m, 1 H, Ph-H), 6.64 (d, J=16.0 Hz, 1H, ═CH), 6.34 (dt, J=16.0 and 5.7 Hz, 1 H, ═CH), 6.16 (t, 1 H, J=6.7 Hz,H-1′), 5.24 (d, J=3.9 Hz, 1 H, D₂O exchangeable, 3′-OH), 5.04 (t, J=4.9Hz, 1 H, D₂O exchangeable, 5′-OH), 4.22 (m, 1 H, H-3′), 4.18 (s, 2 H,5-CH₂a and 5-CH₂b), 4.11 (d, J=5.7 Hz, 2 H, CH₂), 3.77 (m, 1 H, H-4′),3.57 (m, 2 H, H-5′a and H-5′b), 2.09 (dd, J=6.5 and 4.9 Hz, 2 H, H-2′aand H-2′b).

5-(3-Phenyl-2-propenyloxy)methyl-2′-deoxyuridine-5′-triphosphate: Thiscompound has not been made, but it may be synthesized according thefollowing method, or a modified version thereof: POCl₃ (9 μL, 0.1 mmol)could be added to a solution of compound dU.n14 (28 mg, 0.066 mmol) andproton sponge (28 mg, 0.13 mmol) in trimethylphosphate (0.4 mL), and thereaction could then be stirred at 0° C. under a nitrogen atmosphere fortwo hours. A solution of bis-tri-n-butylammonium pyrophosphate (285 mg,0.6 mmol) and tri-n-butylamine (120 μL) in anhydrous DMF (1.2 mL) couldthen be added. After 30 minutes of stirring, triethylammoniumbicarbonate buffer (1 M, pH 7.5; 10 mL) could be added. The reactioncould then be stirred for one hour at room temperature and thenconcentrated in vacuo. The residue could then be dissolved in a mixtureof water (6 mL) and acetonitrile (4 mL), filtered, and purified by anionexchange chromatography using a Q Sepharose FF column (2.5×10 cm) with alinear gradient of 25% acetonitrile/75% triethylammonium bicarbonate(TEAB, 0.1M) to 25% acetonitrile/75% TEAB (1.5 M) over 240 min at 4.5ml/min. The fractions containing triphosphate could then be combined andlyophilized to yield5-(3-phenyl-2-propenyloxy)methyl-2′-deoxyuridine-5′-triphosphateWW6p014.

Example 10 Chemical Cleavage Results of dU Analogs Chemical cleavage of5-(benzyloxy)methyl-2′-deoxyuridine

Chemical Cleavage Using Heterogenous Palladium Catalyst: To a solutionof 5-(benzyloxy)methyl-2′-deoxyuridine (29 mg, 0.082 mmol) in absoluteethanol (2 mL) under a nitrogen atmosphere was added 10 mg of palladiumon activated carbon (10 wt. %, 10 mg) (CAUTION: flammable solid! Ensurehandling in oxygen-free atmosphere). The mixture was flushed withhydrogen gas and stirred at room temperature for 30 minutes while beingmonitored by TLC every five minutes. The mixture was filtered,concentrated under reduced pressure, and dried under vacuum to yieldthymidine (18 mg, 88%) that was identified by comparison to theauthentic sample (TLC and ¹H NMR).

Chemical Cleavage Using Homogenous Palladium Catalyst: To a solution of5-(benzyloxy)methyl-2′-deoxyuridine (3.48 mg, 0.01 mmol) in absoluteethanol (0.1 mL) under a nitrogen atmosphere a solution of sodiumtetrachloropalladate (II) (0.6 mg, 0.002 mmol) in absolute ethanol (0.9mL) was added. The mixture was flushed with hydrogen gas and stirred atroom temperature while being monitored by TLC every five minutes. After20 minutes TLC indicated complete disappearance of starting material.The sole cleavage product was identified to be thymidine by comparisonto the authentic sample on TLC.

Chemical cleavage of 5-(2-isopropylbenzyloxy)methyl-2′-deoxyuridine

Chemical Cleavage Using Heterogenous Palladium Catalyst: To a solutionof 5-(2-isopropylbenzyloxy)methyl-2′-deoxyuridine in absolute ethanol(10 mM, 1 mL) under a nitrogen atmosphere was added of palladium onactivated carbon (10 wt. %, 2 mg) (CAUTION: flammable solid! Ensurehandling in oxygen-free atmosphere). The mixture was flushed withhydrogen gas and stirred at room temperature for five minutes whilebeing monitored by TLC every one minute. After five minutes TLCindicated complete disappearance of starting material. The sole cleavageproduct was identified to be thymidine by comparison to the authenticsample on TLC.

Chemical cleavage of 5-(2-phenylbenzyloxy)methyl-2′-deoxyuridine

Chemical Cleavage Using Heterogenous Palladium Catalyst: To solution of5-(2-phenylbenzyloxy)methyl-2′-deoxyuridine in absolute ethanol (10 mM,1 mL) under a nitrogen atmosphere was added palladium on activatedcarbon (10 wt. %, 2 mg) (CAUTION: flammable solid! Ensure handling inoxygen-free atmosphere). The mixture was flushed with hydrogen gas andstirred at room temperature for five minutes while being monitored byTLC every 1 minute. After five minutes TLC indicated completedisappearance of starting material. The sole cleavage product wasidentified to be thymidine by comparison to the authentic sample on TLC.

Chemical Cleavage of 5-(2,6-dimethylbenzyloxy)methyl-2′-deoxyuridine

Chemical Cleavage Using Heterogenous Palladium Catalyst: To a solutionof 5-(2,6-dimethylbenzyloxy)methyl-2′-deoxyuridine in absolute ethanol(10 mM, 1 mL) under a nitrogen atmosphere was added palladium onactivated carbon (10 wt. %, 2 mg) (CAUTION: flammable solid! Ensurehandling in oxygen-free atmosphere). The mixture was flushed withhydrogen gas and stirred at room temperature for five minutes whilebeing monitored by TLC every 1 minute. After five minutes TLC indicatedcomplete disappearance of starting material. The sole cleavage productwas identified to be thymidine by comparison to the authentic sample onTLC.

Chemical cleavage of 5-(benzyloxy)methyl-2′-deoxyuridine

To a solution of 5-benzyloxy-2′-deoxyuridine (10 mg) in ethanol (1 mL)was added Pd/C (10%, 10 mg), and the mixture was stirred for fiveminutes. Hydrogen was introduced to the system via a balloon and thereaction mixture was stirred at room temperature. Aliquots (5 μL) weretaken out from the reaction mixture at various time point intervals andwere analyzed by thin layer chromatography and HPLC. Complete cleavageto thymidine was observed after ten minutes at room temperature (Scheme42 and FIG. 4). The intermediate of the cleavage was identified to be5-hydroxymethyl-dU generated from initial removal of benzyl group, andHOMedU was further reduced to thymidine.

Cleavage of 5-(1-phenyl-2-methyl-propyloxy)methyl-2′-deoxyuridine usingcatalytic hydrogenolysis: To a solution of5-(1-phenyl-2-methyl-propyloxy)methyl-2′-deoxyuridine (13 mg) in ethanol(3 mL) was added Pd/C (10%, 15 mg) and the mixture was stirred for fiveminutes. Hydrogen was introduced to the system via a balloon and thereaction mixture was stirred at room temperature. Aliquots (5 μL) weretaken out from the reaction mixture various time point intervals andwere analyzed by thin layer chromatography and HPLC. Complete cleavageto thymidine was observed after 240 minutes at room temperature (Scheme43 and FIG. 5). The slow cleavage of the α-isopropyl substituted5-benzyloxymethyl-dU may be caused by the steric hindrance presented bythe substitution when the compound binds to the catalyst surface. Thus,the rate of chemical cleavage is substantially reduced for5-benzyloxymethyluridine analogs when substituted of the α-carbon, whichotherwise is important for termination and discrimination properties ofthese compounds. Without being bound by theory substitution of the2-position of the benzyl ring, but not the α-carbon position can alsoaffect termination properties (see Table 2), which provide fastercleaving nucleotides.

* * *

All of the methods disclosed and claimed herein can be made and executedwithout undue experimentation in light of the present disclosure. Whilethe compositions and methods of this invention have been described interms of preferred embodiments, it will be apparent to those of skill inthe art that variations may be applied to the methods and in the stepsor in the sequence of steps of the method described herein withoutdeparting from the concept, spirit and scope of the invention. Morespecifically, it will be apparent that certain agents which are bothchemically and physiologically related may be substituted for the agentsdescribed herein while the same or similar results would be achieved.All such similar substitutes and modifications apparent to those skilledin the art are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

REFERENCES

The following references, and those listed in the Appendix, to theextent that they provide exemplary procedural or other detailssupplementary to those set forth herein, are specifically incorporatedherein by reference.

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What is claimed is:
 1. A method of identifying a plurality of bases in atarget nucleic acid, comprising the following steps: (i) attaching the5′-end of a primer to a solid surface; (ii) hybridizing a target nucleicacid to the primer attached to the solid surface; (iii) adding acompound of the formula:

wherein: Z is —O—, —S—, —NH—, —OC(O)O—, —NHC(O)O—, —OC(O)NH— or—NHC(O)NH—; R₁ is triphosphate or α-thiotriphosphate; R₂ is hydrogen orhydroxy; R₃ and R₄ are each independently: hydrogen, hydroxy, halo oramino; or alkyl_((C≦12)), alkenyl_((C≦12)), alkynyl_((C≦12)),aryl_((C≦12)), aralkyl_((C≦12)), heteroaryl_((C≦12)),heteroaralkyl_((C≦12)), alkoxy_((C≦12)), aryloxy_((C≦12)),aralkoxy_((C≦12)), hetero-aryloxy_((C≦12)), heteroaralkoxy_((C≦12)),alkylamino_((C≦12)), dialkylamino_((C≦12)), arylamino_((C≦12)),aralkyl-amino_((C≦12)), or a substituted version of any of these groups;or R₅, R₇, and R₈ are each independently: hydrogen, hydroxy, halo,amino, nitro, cyano or mercapto; alkyl_((C≦12)), alkenyl_((C≦12)),alkynyl_((C≦12)), aryl_((C≦12)), aralkyl_((C≦12)), heteroaryl_((C≦12)),heteroaralkyl_((C≦12)), acyl_((C≦12)), alkoxy_((C≦12)),alkenyloxy_((C≦12)), alkynyl-oxy_((C≦12)), aryloxy_((C≦12)),aralkoxy_((C≦12)), heteroaryl-oxy_((C≦12)), heteroaralkoxy_((C≦12)),acyloxy_((C≦12)), alkylamino_((C≦12)), dialkylamino_((C≦12)),alkoxy-amino_((C≦12)), alkenylamino_((C≦12)), alkynylamino_((C≦12)),arylamino_((C≦12)), aralkylamino_((C≦12)), heteroaryl-amino_((C≦12)),heteroaralkylamino_((C≦12)), alkyl-sulphonylamino_((C≦12)),amido_((C≦12)), alkylthio_((C≦12)), alkenylthio_((C≦12)),alkynylthio_((C≦12)), arylthio_((C≦12)), aralkylthio_((C≦12)),heteroarylthio_((C≦12)), heteroaralkyl-thio_((C≦12)), acylthio_((C≦12)),thioacyl_((C≦12)), alkyl-sulfonyl_((C≦12)), arylsulfonyl_((C≦12)),alkyl-ammonium_((C≦12)), alkylsulfonium_((C≦12)), alkyl-silyl_((C≦12)),or a substituted version of any of these groups; R₆ is a-linker-reporter; or a salt thereof; (iv) adding a nucleic acidreplicating enzyme to the hybridized primer/target nucleic acid complexto incorporate the compound of step (iii) into the growing primerstrand, wherein the incorporated compound of step (iii) terminates thepolymerase reaction at an efficiency of between about 70% to about 100%;(v) washing the solid surface to remove unincorporated components; (vi)detecting the reporter group of the incorporated compound of step (iii);(vii) a photochemical cleavage step to remove the terminating moiety ofthe incorporated compound, wherein the terminating moiety has theformula:

(viii) washing the solid surface to remove the cleaved terminatingmoiety; and (ix) repeating steps (iii) through (viii) one or more timesto identify the plurality of bases in the target nucleic acid.
 2. Amethod of identifying a plurality of bases in a target nucleic acid,comprising the following steps: (i) attaching the 5′-end of a targetnucleic acid to a solid surface; (ii) hybridizing a primer to the targetnucleic acid attached to the solid surface; (iii) adding a compound ofthe formula:

wherein: Z is —O—, —S—, —NH—, —OC(O)O—, —NHC(O)O—, —OC(O)NH— or—NHC(O)NH—; R₁ is triphosphate or α-thiotriphosphate; R₂ is hydrogen orhydroxy; R₃ and R₄ are each independently: hydrogen, hydroxy, halo oramino; or alkyl_((C≦12)), alkenyl_((C≦12)), alkynyl_((C≦12)),aryl_((C≦12)), aralkyl_((C≦12)), heteroaryl_((C≦12)),heteroaralkyl_((C≦12)), alkoxy_((C≦12)), aryloxy_((C≦12)),aralkoxy_((C≦12)), hetero-aryloxy_((C≦12)), heteroaralkoxy_((C≦12)),alkylamino_((C≦12)), dialkylamino_((C≦12)), arylamino_((C≦12)),aralkyl-amino_((C≦12)), or a substituted version of any of these groups;or R₅, R₇, and R₈ are each independently: hydrogen, hydroxy, halo,amino, nitro, cyano or mercapto; alkyl_((C≦12)), alkenyl_((C≦12)),alkynyl_((C≦12)), aryl_((C≦12)), aralkyl_((C≦12)), heteroaryl_((C≦12)),heteroaralkyl_((C≦12)), acyl_((C≦12)), alkoxy_((C≦12)),alkenyloxy_((C≦12)), alkynyl-oxy_((C≦12)), aryloxy_((C≦12)),aralkoxy_((C≦12)), heteroaryl-oxy_((C≦12)), heteroaralkoxy_((C≦12)),acyloxy_((C≦12)), alkylamino_((C≦12)), dialkylamino_((C≦12)),alkoxy-amino_((C≦12)), alkenylamino_((C≦12)), alkynylamino_((C≦12)),arylamino_((C≦12)), aralkylamino_((C≦12)), heteroaryl-amino_((C≦12)),heteroaralkylamino_((C≦12)), alkyl-sulphonylamino_((C≦12)),amido_((C≦12)), alkylthio_((C≦12)), alkenylthio_((C≦12)),alkynylthio_((C≦12)), arylthio_((C≦12)), aralkylthio_((C≦12)),heteroarylthio_((C≦12)), heteroaralkyl-thio_((C≦12)), acylthio_((C≦12)),thioacyl_((C≦12)), alkyl-sulfonyl_((C≦12)), arylsulfonyl_((C≦12)),alkyl-ammonium_((C≦12)), alkylsulfonium_((C≦12)), alkyl-silyl_((C≦12)),or a substituted version of any of these groups; R₆ is a-linker-reporter; or a salt thereof; (iv) adding a nucleic acidreplicating enzyme to the hybridized primer/target nucleic acid complexto incorporate the compound of step (iii) into the growing primerstrand, wherein the incorporated compound of step (iii) terminates thepolymerase reaction at an efficiency of between about 70% to about 100%;(v) washing the solid surface to remove unincorporated components; (vi)detecting the reporter group of the incorporated compound of step (iii);(vii) optionally adding one or more chemical compounds to permanentlycap unextended primers; (viii) a photochemical cleavage step to removethe terminating moiety of the incorporated compound, wherein theterminating moiety has the formula:

(ix) washing the solid surface to remove the cleaved terminating moiety;and (x) repeating steps (iii) through (ix) one or more times to identifythe plurality of bases in the target nucleic acid.
 3. The method ofclaim 1, wherein the compound is incorporated by a nucleic acidreplicating enzyme that is a DNA polymerase.
 4. The method of claim 1,wherein the photo-cleavage is performed using a wavelength of lightranging between 300 nm to 400 nm.
 5. The method of claim 1, wherein Z is—O—.
 6. The method of claim 1, wherein R₁ is a triphosphate.
 7. Themethod of claim 1, wherein R₁ is α-thiotriphosphate.
 8. The method ofclaim 1, wherein R₂ is hydrogen.
 9. The method of claim 1, wherein R₃ isalkyl_((C≦12)) or a substituted version thereof.
 10. The method of claim1, wherein R₃ is selected from the group consisting of methyl, ethyl,n-propyl, isopropyl and tert-butyl.
 11. The method of claim 1, whereinR₃ is tert-butyl.
 12. The method of claim 1, wherein R₄ is hydrogen. 13.The method of claim 1, wherein R₅ is hydrogen.
 14. The method of claim1, wherein the linker of the -linker-reporter is:

wherein X is —O—, —S—, or —NH—; or alkanediyl_((C≦12)),alkenediyl_((C≦12)), alkynediyl_((C≦12)), arenediyl_((C≦12)),heteroarenediyl_((C≦12)), or a substituted version of any of thesegroups; and n is an integer from 0-12.
 15. The method of claim 14,wherein X is —C≡C—.
 16. The method of claim 14, wherein n is zero. 17.The method of claim 14, wherein the reporter is:


18. The method of claim 1, wherein R₇ is hydrogen.
 19. The method ofclaim 1, wherein R₈ is hydrogen.